Cardiac stress produced by hypertension or excess volume loading results in different types of hypertrophy. Elevated left ventricular pressure rapidly results in increased myocardial protein synthesis in vivo and in vitro, but such rapid alterations are not consistently seen in volume loading. The difference in response is difficult to clarify since it is not possible to effect alterations in left ventricular pressure or perfusion without profoundly affecting coronary perfusion. The present study describes cardiac protein synthesis in the right ventricle of the young guinea pig heart in vitro by utilizing a perfusion model in which the right ventricle could be stressed by elevations of pressure or volume loading in the presence of constant and restricted coronary perfusion. With coronary flow maintained at 4 ml/min per heart equivalent to 25 ml/min/g dry wt, an increase in right ventricular pressure from normal levels of 3 mm Hg to 11 mm Hg resulted in a 60 percent increase of myocardial incorporation of (14C)lysine into protein. However, with further increases of right ventricular pressure to 22 mm Hg, protein synthesis dropped back to normal levels. The falloff in protein synthesis was not due to decreased contractility, alterations in intracellular lysine pool specific activity, or alterations in distribution of coronary flow. a 60 percent increase in coronary perfusion was again associated with a similar response of protein synthesis to progressive elevations of pressure despite a rise in the ATP levels and a fall in lactate production. Thus, a deficiency of O2 did not entirely explain the decline of protein synthesis with maximal pressures. At all levels of coronary perfusion, volume loading for 3 h did not result in increased protein incorporation of (14C)lysine. The studies support a relationship between ventricular pressure and protein synthesis unrelated to coronary flow per se. A pressure receptor triggering protein synthesis within the ventricular wall is postulated. Such a relationship is not apparent in short-term volume loading in vitro.
S S Schreiber, M A Rothschild, C Evans, F Reff, M Oratz
The mode of action of the hypocholesteremic drug neomycin (2 g/day) was studied in four patients. All showed a significant reduction in plasma cholesterol concentrations (mean 25 percent, range 18-31 percent), and in one of three patients with hyperglyceridemia there was a decrease of plasma triglycerides of 26 percent. Cholesterol absorption was measured in three of four patients: there was a marked decrease. Sterol balance studies in four patients showed an unabating increase in fecal neutral steroid excretion (mean increase 345 mg/day, range 323-361) for 3-5 wk after plasma cholesterol levels had reached a new and lower plateau. Fecal acidic steroid excretion increased temporarily in two patients, with a sustained increase of 93 mg/day in only one. Daily stool weights increased significantly in three of four patients, though none had steatorrhea; there was a significant reduction in excretion of secondary bile acids; neutral sterol degradation rates were not affected by the drug. Slopes of plasma cholesterol-specific activity time curves did not change. These results fail to support the suggestion that neomycin acts as a bile acid precipitant. The finding of increased fecal neutral steroid excretion is consistent with decreased cholesterol absorption, but also with increased cholesterol absorption, but also with increased cholesterol synthesis (secondary to release of negative feedback control), with increased flux of cholesterol from tissues, or with a combination of all three actions.
A Sedaghat, P Samuel, J R Crouse, E H Ahrens Jr
The mechanisms and kinetics of the immunosuppressive effects of alternate-day prednisone were investigated in a group of patients with a variety of inflammatory diseases receiving a range of alternate-day prednisone doses from 5 to 120 mg. Total circulating lymphocyte and monocyte counts, as well as proportions of lymphocyte subpopulations defined both by surface markers and by in vitro functional capacities, were studied. At 8 a. m. of the day on prednisone, just before drug administration, lymphocyte and monocyte counts, proportions of lymphocyte subpopulations, as well as in vitro lymphocyte blastogenic responses to various mitogenic and antigenic stimuli were normal. 4 h after the administration of prednisone, there was a profound lymphocytopenia and monocytopenia, with a differential depletion of thymus-derived lymphocytes as well as various functionally defined lymphocyte subpopulations. Lymphocyte kinetic studies using a radioactive chromium-labeled autologous lymphocytes showed that the lymphocytopenia was due predominantly to a transient depletion of the recirculating portion of the intravascular lymphocytepool. All these parameters returned to normal by 8 a.m. of the following day (off prednisone) and remained normal throughout the day. This very transient lymphocytopenia and monocytopenia after prednisone, with normal cell numbers, proportions, and functions throughout the remainder of the 2-day cycle, was associated with suppression of disease activity, yet did not affect cutaneous delayed hypersensitivity in these patients nor increase the likelihood of infectious complications. This drug-associated cyclic and transient monocytopenia and selective lymphocytopenia is best explained by a redistribution of recirculating lymphocytes to other body compartments, particularly the bone marrow.
A S Fauci, D C Dale
Recently we reported that rapid killing of Escherichia coli by granulocytes or granulocyte fractions is accompanied by an equally rapid and discrete increase in permeability of the microbial envelope (Beckerdite, Mooney, Weiss, Franson, and Elsbach. 1974. J. Exp. Med. 140: 396-409). Most of this permeability-increasing activity (PI) is found in a crude granule preparation. PI is quantitatively recovered in a 23,000-g supernatant fraction (Sup II) after sulfuric acid extraction of granulocyte homogenates prepared in water. PI is nondialyzable, destroyed by pronase and trypsin, stable at 4degreesC for at least 2 mo, and destroyed by heating at 94degreesC. Anionic substances, such as heparin sulfate and isolated E. coli lipopolysaccharide, bind to and inhibit PI. PI has been purified up to 1,000-fold from homogenate in a yield of 50percent by acid extraction and carboxymethyl-Sephadex chromatography. Such purified fractions have bactericidal activity that equals that of disrupted granulocytes and Sup II, are similarly enriched with respect to granule-associated phospholipase, and protease activities. Whereas E. coli, sensitive to PI, binds or inactivates solubilized PI, a resistant strain of Serratia marcescens does not. Binding of PI to sensitive microorganisms seems to be necessary for expression of its biological activity since both the apparent binding to and the biological effect of PI on E. coli are completely blocked by 10-20 mM Mg2+ or Ca2+. Mg2+ or Ca2+ can reverse the effect on E. coli permeability produced by Sup II or the carboxymethyl-Sephadex fraction but not that produced by granulocyte homogenate. The close association of bactericidal, phospholipase A2, and permeability-increasing activities towards several gram-negative bacterial species suggests that they may be related.
J Weiss, R C Franson, S Beckerdite, K Schmeidler, P Elsbach
Regional myocardial blood flow was measured in nine dogs at rest and during three levels of treadmill exercise by using left atrial injections of 7-10-mum radioactive microspheres. At rest, heart rate was 76 plus or minus 3 beats/min (mean plus or minus SEM), mean left ventricular myocardial flow was 0.94 plus or minus 0.09 ml/min/g and endocardial flow (endo) exceeded epicardial flow (epi) in all regions (endo/epi equals 1.12-1.33). When treadmill exercise was regulated to increase heart rates from 152 plus or minus 3 to 190 plus or minus 3 to 240 plus or minus 6 beats/min, myocardial blood flow (MBF) to all regions of the left ventricle increased linearly with heart rate (HR) from 1.83 plus or minus 0.11 to 2.75 plus or minus 0.22 to 3.90 plus or minus 0.26 ml/min/g (MBF EQUALs 0.0175 HR - 0.523 PLUS OR MINUS 0.614, R EQUALS 0.87). Exercise abolished the gradient of blood flow favoring the left ventricular endocardium at rest, so that the endo/epi flow ratios were not significantly different from 1.00. Right ventricular flows were consistently less than corresponding left ventricular flows, but showed a similar linear increase with heart rate. Right ventricular endo/epi ratios were not different from 1.00 either at rest or during exercise. Thus, exercise resulted in increased myocardial blood flow to all regions of the left and right ventricles with maintenance of subendocardial flow equal to subepicardial flow.
R M Ball, R J Bache, F R Cobb, J C Greenfield Jr
Isolated hepatic nuclei from euthyroid rats were incubated with tracer (125I)L-triiodothyronine (T3) and increasing doses of nonradioactive T3 for 30 min at 37degrees C. The T3 bound specifically to nuclear sites increased with increasing T3 doses to a plateau, which represented the nuclear binding capacity, M. Addition of 1 mM KCN, NaF, dinitrophenol, oriodoacetate did not affect nuclear binding, indicating that active metabolism was not required. Kinetic studies showed that the nuclear sites were equilibrated with T3 within 30 min of incubation (one-half maximal binding at 3 min) and that the rate of release of T3 in vitro (0.058 min-1) was the same for endogenous T3 or for T3 bound to nuclei in vitro. Nuclear T3 resisted extraction with 0.14 M NaC1 buffered at pH 7.5, but both endogenous hormone and T3 bound in vitro were readily extracted by 0.4 M KC1 at pH 8.0. The elution profiles of endogenous and in vitro-bound T3 from Sephadex G-100 columns showed a common protein peak with a molecular weight of 60-65,000, assuming a globular protein. Scatchard analysis of in vitro displacement studies showed a single class of binding sites. Mean M equals 0.23 times 10-9 M or 0.85 ng T3 for nuclei isolated from 1 g of liver. Mean M closely corresponded to that anticipated from reported in vivo studies. The apparent association constant Ka for the nuclear sites, 5.55 times 108 M-1, was lower than in studies in vivo, probably attributable to the different ionic milieu of nuclei in the incubation buffer and in the intact cell. Thus, the identity of the nuclear T3 binding sites studied in vitro to those reported for endogenous hormone is demonstrated by similar binding capacities, release rates, analogue binding affinities (previously reported), and localization to chromatin nonhistone proteins of comparable molecular weight. The role of cytosol protein in nuclear binding was assessed by comparing binding parameters for extensively washed nuclei and nuclei incubated either with contaminating or added cytosol. No difference in Ka or M was found. Moreover, it was unlikely that specific cytosol proteins were already present in nuclei and functioned during incubation as a shuttle for T3, since Ka and M for nuclei obtained from athyreotic rats were similar to Ka and M for nuclei from euthyroid animals. Thus, an initial interaction between T3 and specific cytosol proteins does not appear to be a prerequisite for translocation of T3 to nuclear sites.
M I Surks, D H Koerner, J H Oppenheimer
Isometric performance at 29degreesC was measured in left ventricular trabeculae carneae from young adult (6-mo) and aged (25-mo) rats (n equals 18 in each group). Active tension and maximal rate of tension development did not differ with age, but contraction duration was 255plus or minus6 ms in the young adult and 283plus or minus6 ms in the aged group (P less than0.001). Although catecholamine content per gram heart weight was less in the aged myocardium, additional experiments showed that neither 1 times 10-6 M propranolol nor pretreatment with 6-hydroxydopamine eliminated the age difference in contraction duration. To determine if this age difference resulted from a prolonged active state, electromechanical dissociation and the overshoot of contraction duration during recovery from hypoxia were measured. During paired stimulation greater mechanical refractoriness was found in aged muscles (P less than0.01), but intracellular action potential recordings showed no age difference in the electrical refractory period. On recovery from hypoxia, contraction duration overshoot was 117plus or minus 4percent of control in the young and 138plus or minus 4percent of control in the aged muscles (P less than0.01). The greater electromechanical dissociation and greater overshoot in contraction duration following hypoxia in aged myocardium suggests that prolonged contraction duration in aged myocardium results from a prolonged active state rather than changes in passive properties or myocardial catecholamine content.
E G Lakatta, G Gerstenblith, C S Angell, N W Shock, M L Weisfeldt
Previous studies have demonstrated an increase in blood flow to extremities involved by Paget's disease of bone. It has been assumed that the increase in blood flow is through bone, but warmth of skin overlying Pagetic bone suggests that cutaneous blood flow might be increased. In three patients with Paget's disease involving one extremity, we compared blood flow in "Pagetic" extremities with flow in the contralateral normal extremities. Resting blood flow (measured with water plethysmographs) was 14.2plus or minus2.9 (meanplus or minusSE) ml/min times 100 ml extremity in the Pagetic limbs. The contribution of cutaneous flow to the increase in extremity blood flow was evaluated with epinephrine iontophoresis, which suppresses flow to skin but not to underlying tissue. Epinephrine iontophoresis of the Pagetic extremities decreased blood flow from 14.2plus or minus2.9 to 3.6plus or minus1.5 ml/min. Local heating (which increases cutaneous flow only) failed to increase blood flow in the Pagetic extremities as much as it did in the normal extremities. This suggests that cutaneous vessels in the Pagetic extremities were already dilated. During heating, blood flow in the normal extremities was similar to resting flow in the Pagetic extremities; this indicates that increases in cutaneous flow could account for most of the increase in total blood flow in the Pagetic extremities. Adrenergic control of blood flow to the Pagetic extremities was compared with that of the normal extremities. Vasoconstrictor responses to reflex stimuli in the Pagetic extremities were reduced; when vasoconstriction occurred it was gradual and sustained after termination of the stimuli, which suggests an exaggerated humoral response but reduced neural response to the stimuli. Intravenous epinephrine produced vasoconstriction in the Pagetic extremities and vasodilatation in the normal extremities. In summary, responses to epinephrine iontophoresis and heating suggest that the increase in blood flow in Pagetic extremities is primarily the result of cutaneous vasodilatation.
D D Heistad, F M Abboud, P G Schmid, A L Mark, W R Wilson
The detection of platelet isoantibodies by the release of (3H)serotonin from platelets has been evaluated. The conditions for optimal release of (3H)serotonin with platelet isoantibodies using a microtechnique have been defined. A group of cardiac surgery patients were followed pre- and post-transfusions, with 48percent developing a positive serotonin release assay. Of these patients, 16percent also had a platelet complement-fixing and/or lymphocytotoxic isoantibody. There was variation in the degree of correlation between (3H)serotonin release and lymphocytotoxicity using individual National Institutes of Health typing serum. The matching obtained between family members by both techniques showed a close correlation when each technique was evaluated separately using the same NIH typing serum. The detection of iso-antibodies in patients with hematological malignancies correlated with the unresponsiveness to unmatched platelet transfusions in 15 out of 17 cases. The use of the patient's isoantibody to matched platelets of family members by (3H)serotonin release correlated well with the clinical response to transfusion with these platelets. The data suggest that (a) platelet isoantibodies can be detected with increased frequency by (3H)serotonin release; (b) (3H)serotonin release is a specific reaction depending on the surface antigen of the platelet; and (c) the method can be used to match compatible family members for platelet transfusions.
J P Gockerman, R P Bowman, M E Conrad
The phenomenon of redistribution of surface membrane immunoglobulin (Ig) components (capping) has been well described in mouse lymphoid cells. The characteristics of this process in human lymphocytes are less clear. This study characterizes the phenomenon of surface membrane Ig redistribution of normal and chronic lymphocytic leukemia (CLL) lymphocytes with the use of fluoroscein-labeled anti-Ig sera. Normal lymphocytes underwent rapid cap formation after incubation with anti-Ig serum in the cold and subsequent rewarming. The morphology was characteristic with aggregation over the pole of the cell opposite the nucleus and over the uropod when present. The process was energy dependent but independent of protein synthesis, and could be inhibited by vincristine, vinblastine, and colchicine but not by cytochalasin B. CLL cells, on the other hand, though showing fluorescent complex aggregation on the surface, rarely demonstrated unidirectional movement of these aggregates to form a cap. Cap formation in these cells could not be stimulated by supplementing the energy source or protein concentration of the medium nor by adding glutamic acid which could partially reverse the vincristine and vinblastine inhibition of normal capping. The failure of agents which inhibit motility to inhibit capping of the normal lymphocytes suggests that active locomotion is not a direct prerequisite for capping. The results also suggest the involvement of microtubules in normal capping and the possibility that abnormal membrane structure or microtubular function could explain the failure of CLL cells to behave normally in this regard. The role of this cellular defect in the immune deficiencies exhibited by many patients with CLL, however, is not established.
H J Cohen
Intravenous hyperalimentation was done in 11 underweight adults whose body weight (body wt) was less than 85 percent of ideal. For the first 6 days, "complete formula" was infused furnishing per kilogram ideal body wt per day: 15 g glucose, 0.40 g N, 0.018 g P, 2.4 meq K, 3.0 meq Na, 2.3 meq C1, 0.5 meq Mg, 0.45 meq Ca, and 50 ml H20. Patients gained weight at an average rate of 9.0 g/kg ideal body wt/day and showed average balances/kilogram ideal body wt/day as follows: plus 0.14 g N; plus 0.012 g P; plus 0.43 meq K; plus 0.49 meq Na; plus 0.37 meq Cl; and plus 0.085 meq Ca. Application of standard equations to the elemental balances indicated weight gain consisted of 35-50 percent protoplasm, 35-50 percent extracellular fluid, 5-25 percent adipose tissus, and less than 1 percent bone. Withdrawas of N, P, Na, or K impaired or abolished retention of other elements. Removal of N halted retention P, K, Na and C1; withdrawal of K stopped retention of N and P; and removal of Na or P interrupted retention of all other elements. Weight gain continued at a rate of 1.4-3.1 g/kg ideal body wt/day despite zero or negative elemental balances of N, K, P, and sometimes Na and C1. Calculations showed that weight gain during infusion of fluids lacking N, P, K, or Na consisted largely of adipose tissue, with little or no contribution by protoplasm or extracellular fluid. Data show that repletion of protoplasm and extracellular fluid of wasted adults by intravenous hyperalimentation is retarded or abolished if N, P, Na, or K is lacking. Repletion of bone mineral does not occur in absence of Na or P but proceeds in absence of N, P, K, or Na. Thus, quality of weight gained by underfed adult patients during hyperalimentation depends on elemental composition of the infusate.
D Rudman, W J Millikan, T J Richardson, T J Bixler 2nd, J Stackhouse, W C McGarrity
The role of bile canalicular and microsomal membranes in the synthesis and transport of biliary lipids was investigated by using the isolated perfused rat liver model. Labeled lecithin precursors ((3H)-palmitic acid, (14C)linoleic acid, (3H)choline, and 32PO4) and a cholesterol precursor ((3H)mevalonic acid) were administered with and without sodium taurocholate. The incorporation pattern of these labeled precursors into linoleyl and arachidonyl lecithins and cholesterol fractions of microsomes, bile canaliculi, and bile were examined at 30-min intervals up to 90 min. Marker enzymes and electron microscopy indicated that isolated subfractions of plasma membranes were enriched with bile canaliculi (less than 10 percent microsomal contamination). Taurocholate significantly stimulated the incorporation of 32PO4, (3H)choline, (3H)palmitic acid, and (14C)linoleic acid into linoleyl and arachidonyl lecithin with parallel incorporation curves for microsomal and bile canalicular membranes throughout the 90-min study period. During the 30-60-min period, however, these same lecithin fractions in bile significantly exceeded the specific activity of the membrane lecithins. The enzyme CDP-choline diglyceride transferase was virtually absent from canaliculi relative to microsomes, indicating that canaliculi lack the capacity for de novo lecithin synthesis. Incorporation of (3H)mevalonic acid into membranous and biliary cholesterol followed a pattern similar to that for lecithin. These data provide evidence that (a) biliary lecithin and cholesterol are derived from a microsomal subpool regulated by the flux of enterohepatic bile acids, (b) the role of the bile canalicular membranes with respect to biliary lipids is primarily transport rather than synthesis, and (c) lecithin and cholesterol are transported together from microsomes to bile. The findings are consistent with the existence of a cytoplasmic lipid complex within the hepatocyte which is actively involved in the intermembrane transport of biliary lipid.
D H Gregory, Z R Vlahcevic, P Schatzki, L Swell
Cholesterol-rich membranes are the hallmark of "spur" red cells. Spur cells accumulate cholesterol from cholesterol-rich serum lipoproteins. Previous studies suggested that this added cholesterol is responsible for both the altered morphology and the destruction of spur cells. To examine this process in the absence of other serum factors, cholesterol-lecithin dispersions with varying amounts of unesterified cholesterol (C) relative to phospholipid (P) were prepared, and their influence on normal human red cells was studied. Cholesterol-rich lipid dispersions (C/P mole ration greater 1.0) transferred cholesterol to both red cell membranes and serum lipoproteins, and cholesterol-poor dispersions (C/P mole ration less 1.0) depleted red cells of cholesterol. Changes in membrane cholesterol paralleled changes in membrane surface area, as calculated from osmotic fragility, with a 0.22 percent variation in surface area per 1.0 percent variation in cholesterol content. Cold-induced compression of membrane surface area was increased in cholesterol-poor red cells (C/P equals 0.4), whereas the surface area of cholesterol-rich membranes (C/P equals 1.80) underwent no compression. Although the Na and K permeability of red cells severely depleted of cholesterol was increased, lesser degrees of depletion had no effect, and the permeability of cholesterol-rich cells was normal. However, increasing membrane cholesterol caused a progressive decrease in red cell deformability, as measured by filtration. Cholesterol-poor red cells were spherocytic in appearance and cholesterol-rich cells were broad and flat, indicative of their surface areas. In addition, cholesterol-rich cells had an irregular contour due to folding of the periphery of the cell. This shape abnormality was identical to that of both spur cells after splenectomy and normal red cells incubated in spur serum. Normalization of the C/P of spur serum by added phospholipid prevented the increase in membrane cholesterol and surface area and the transformation of cell shape. These studies establish that the cholesterol content of red cells is dependent on the C/P of their milieu, either lipoproteins or cholesterol-lecithin dispersions. Moreover, the surface area, deformability, and contour of cholesterol-rich red cells are a direct function of their increased membrane C/P. Although cholesterol-rich spur cells are further modified in the circulation of patients with spleens, this abnormality of the membrane lipid bilayer, induced by cholesterol-rich cholesterol-lecithin dispersions, represents the primary spur cell defect.
R A Cooper, E C Arner, J S Wiley, S J Shattil
A radioimmunoassay has been developed for Somatomedin B, a growth hormone-dependent factor that stimulates DNA synthesis in human glia-like cells. The sensitivity permits detection of this factor in human plasma diluted 1: 20,000 and in monkey plasma diluted 1: 5,000. It is not measurable in nonprimate plasma diluted 1: 20. The concentration in growth hormone-deficient adult patients is equivalent to 6.6plus or minus0.5 ug/ml of a highly purified somatomedin preparation. In acromegaly the concentration is 19.3plus or minus2.3 ug/ml and falls after definitive therapy that results in a decrease in plasma growth hormone. In unextracted human plasma the immunoreactive Somatomedin B is associated with a plasma protein at least as large as gamma-globulin and with an electrophoretic mobility on paper resembling the alpha-globulins. The level of Somatomedin B in the bound form in human plasma under steady-state conditions may depend on the rate of production of the peptide and/or the concentration of the plasma-binding protein. At present there is no information concerning which of these is modulated by growth hormone. Immunoreactive Somatomedin B is found predominantly in Cohn plasma fractions III and IV, largely dissociated from the plasma-binding protein. The disappearance curves of labeled purified Somatomedin B and of immunoreactive Somatomedin B from acromegalic plasma administered intravenously to a dog were superposable; the terminal portion of the disappearance curve having a half time of almost an hour.
R S Yalow, K Hall, R Luft
The present study was undertaken to investigate the mechanism of the antilipolytic action of clofibrate (p-chlorophenoxyisobutyrate). Clofibrate, in the dose range of 10-80 mg/199 ml, inhibited the initial rate of norepinephrine-stimulated lipolysis 17-44 percent in isolated rat fat cells. At a dose corresponding to therapeutic levels in vivo (10 mg/100 ml) clofibrate also inhibited hormone-stimulated lipolysis by 20-30 percent in fragments of human subcutaneous fat. Inhibition of lipolysis by clofibrate occurred at all concentrations of norepinephrine and ACTH (0.02-0.1 mug/ml) but did not occur with equilipolytic concentrations of dibutyryl cyclic AMP, suggesting a proximal site of action on the lipolytic sequence. Clofibrate reduced by 60 percent (315plus or minus40 vs. 120plus or minus25 pmol/g lipid; meanplus or minusSEM) the norepinephrine-stimulated initial rise in cyclic AMP, measured 10 min after addition of hormone. Because the antilipolytic effect occurred in the presence of glucose and without altering cellular ATP levels, the reduction in intracellular cyclic AMP levels could not be attributed to uncoupling of oxidative metabolism or to secondary effects of free fatty acid accumulation. In the secondary effects of free fatty acid accumulation. In the presence of procaine-HC1, which blocks hormone-stimulated lipolysis without inhibiting cyclic AMP accumulation, addition of clofibrate prevented the hormone-stimulated rise in cyclic AMP. Clofibrate did not affect the activity of the low-Km 3',5'-cyclic AMP phosphodiesterase in norepinephrine-stimulated adipocytes. These data suggest that the antilipolytic effect of clofibrate is due to its suppression of cyclic AMP production by inhibition of adenylate cyclase. The drug's hypolipidemic action may in part be explained by its antilipolytic effect, which deprives the liver of free fatty acid substrate for lipoprotein synthesis.
M A D'Costa, A Angel
It has been demonstrated that parathyroid hormone (PTH) inhibits the proximal tubular reabsorption of bicarbonate, and increases the urinary excretion of that ion. There is also a qualitative similarity between the alterations of the proximal tubular reabsorption of phosphate, sodium, and water after PTH administration and after acetazolamide administration. These findings suggest that the renal effect of PTH is possibly mediated through the inhibition of carbonic anhydrase in proximal tubules. Therefore, a possible inhibitory effect of PTH on carbonic anhydrase was evaluated in the homogenate of rat renal cortex by an indicator titration method. Incubation of cortical homogenates with PTH for 10 min at 37degreesC inhibited carbonic anhydrase activity. The inhibitory effect of PTH was ATP-, Mg++-, and K+-dependent and temperature-dependent; inactivation of PTH by heating at 100degreesC abolished the effect of PTH both to activate adenylate cyclase and to inhibit carbonic anhydrase. Calcium 5 mM also partially abolished effects of PTH to activate adenylate cyclase and to inhibit carbonic anhydrase. The inhibitory effect of PTH on carbonic anhydrase was specific to renal cortex. Cyclic AMP, the intracellular messenger substance for PTH, also inhibited carbonic anhydrase in renal cortex. The cyclic AMP-induced inhibition was also Mg++ dependent and temperature dependent, and required preincubation at 37degreesC. But 5'-AMP, a metabolic derivative of cyclic AMP without its biological effect, had no inhibitory effect on carbonic anhydrase. All the above results are consistent with the hypothesis that PTH inhibits proximal tubular reabsorption of bicarbonate and phosphate through the inhibition of carbonic anhydrase, and that inhibitory effect is mediated through the cyclic AMP system.
N Beck, K S Kim, M Wolak, B B Davis
Concentrations of IgD and IgE were measured in sera from 165 patients with well-defined immunodeficiency in an effort to find information possibly relevant to the roles of antibodies of these classes in host defense. Values for both immunoglobulins were generally quite low in patients who had marked deficiencies of all three major immunoglobulins, although occasional normal or high normal values for IgD were seen in hypogammaglobulinemic patients. Group mean IgD concentrations were also depressed in patients with Wiskott-Aldrich syndrome and in those with selective IgA deficiency; IgE concentrations were depressed in patients with X-linked immunodeficiency with hyper-IgM and in those with ataxia telangiectasia. IgD and IgE were both significantly elevated in patients with extreme hyperimmunoglobulinemia E and undue susceptibility to infection and in a patient with the Nezelof syndrome; none of these patients had histories suggestive of atopy. In addition, the mean IgE concentration was significantly elevated in patients with selective IgA deficiency, many of whom were atopic, and in those with the Wiskott-Aldrich syndrome. The highest IgD concentration (163 mg/100 ml) was found in serum from a boy with variable immunodeficiency who had a lifelong history of severe recurrent pharyngeal infections, primarily streptococcal in etiology. Recurrent staphylococcal infection was a feature common to many but not all patients with elevated serum IgE concentration. These data may prove useful in the future delineation of biologic roles for antibodies in these two immunoglobulin classes.
R H Buckley, S A Fiscus
With insulin at 0.1 ng/ml, the binding of (125I)insulin in vitro to circulating lymphocytes from 11 obese patients was less than that observed with cells from 10 thin volunteers. Furthermore, with obese cells, unlabeled insulin was less effective in competing with labeled hormone for binding, both at low and high concentrations of unlabeled insulin. These differences were not accounted for by the high concentrations of insulin in the circulation of the obese patients at the time fthe blood was drawn, or by differences in degradation of hormone, or in the characteristics of the cell population. The decrease in binding appears to be due to a lowering of the receptor concentration, but some loss of affinity has not been excluded. Institution of a calorie restricted diet (nine patients) which ameliorated the hyperinsulinemia, produced an improvement in hormone binding. Since the insulin receptors of lymphocytes in metabolic disorders seem to reflect the state of insulin receptors or target cells such as liver and fat, the lymphocytes or other leukocytes appear to be ideal for studies of impaired cell responsiveness to hormones in man.
J A Archer, P Gorden, J Roth
Concanavalin A (con A) is a potent inhibitor of coagulant activity of native tissue factor. Coagulant activity is recovered by addition of alpha-methyl-D-glucoside to inhibited tissue factor. Inclusion of alpha-methyl-D-glucose during incubation of con A with tissue factor preserves coagulant activity. These data suggest that con A interacts reversibly with a carbohydrate residue in such a way as to inhibit coagulant activity of the molecule. Purified tissue factor apoprotein has been recombined with mixed brain phospholipids or purified phospholipids (phosphatidyl ethanolamine or a mixture of phosphatidyl choline with phosphatidyl serine). These preparations were also completely but reversibly inhibited by con A. Thus, purified tissue factor apoprotein appears to donate the affected carbohydrate residue.
F A Pitlick
To determine if pancreatic glucoregulatory hormones can be implicated in the glucose fall of pregnancy, we have measured plasma immunoreactive insulin and glucagon (IRI and IRG) in rats. Fed rats in midgestation show a rise in IRI without a corresponding increase in IRG. In late gestation, IRG rises significantly, but only enough to keep pace with a further rise in IRI. On a molar basis, IRI remains the predominant hormone despite a marked fall in blood glucose. After a 48-h fast IRI falls to comparably low levels in pregnant and virgin rats. A small rise in IRG is seen in virgin but not in pregnant rats despite frank hypoglycemia in the latter. Thus, IRG secretion in pregnancy is diminished relative to IRI in the fed state and fails to increase in the fasted state despite the stimulus of a lower glucose in both instances. To evaluate IRG secretory reserve, the IRG response to i.v. alanine was assessed in late gestation. In fed rats a greater IRG increase is seen in pregnancy; after fasting no difference is seen between pregnant and virgin rats. These results preclude an absolute deficiency in glucagon secretion. Pancreas hormone stores were alos measured in an effort to explain the altered secretory state. We find reciprocal changes in IRI and IRG content favoring IRG in midgestation and IRI in late gestation. Thus, pancreas hormone storage is altered in pregnancy but does not account for the changes in hormone secretion. Rather, pregnancy exerts an effect on the islet secretory process itself. Release of IRI is enhanced relative to IRG regardless of the blood sugar level. These observations suggest that in the pregnant rat circulating levels of insulin and glucagon may act to limit hepatic glucose output. Available evidence from the literature supports the concept of restrained glucose production. It is proposed that a lower blood glucose production. It is proposed that a lower blood glucose in rat pregnancy may be a lesser liability teleologically than would be the obligate nitrogen wasting which accompanies gluconeogenesis.
C D Saudek, M Finkowski, R H Knopp
A variable metric optimization method of numerical analysis has been used to recover known distributions of intrapulmonary ventilation-perfusion ratios from inert gas data. Hypothetical lungs were simulated and corresponding inert gas retentions calculated. By using error-free retentions for seven gases and a 50-compartment model, it was possible to recover distributions containing up to three modes accurately and with greater efficiency than with other numerical methods. When random error of a magnitude consistent with present analytical techniques was introduced into retention data, the recovered distributions differed qualitatively from the original ones. This resulted from the ill-conditioned nature of the mathematical problem, which makes a recovered distribution extremely sensitive to small errors in retention. Thus, present levels of measurement error represent an important limitation in current techniques for deriving distributions from inert gas measurements.
S A Jaliwala, R E Mates, F J Klocke
Studies were performed in pregnant rabbits to assess the effect of inhibition of prostaglandin synthesis on uterine blood flow. Cardiac output and uteroplacental blood flow (UPBF) were measured using radiolabeled microspheres. Prostaglandin E (PGE) concentration was measured by radioimmunoassay in the uterine vein and peripheral artery of the pregnant nephrectomized rabbit. Either meclofenamate or indomethacin 2 mg/kg were utilized to inhibit prostaglandin synthesis. Systemic arterial pressure increased from 86 mm Hg to 98 mm Hg (P less than0.0001) after prostaglandin inhibition. Cardiac output was unchanged after the inhibition of prostaglandin synthesis, 326 ml/min to 7.8 ml/min. Uterine vein PGE concentration was extremely high, 172.4 ng/ml, with concomitant peripheral arterial PGE 2.1 NG/ML. Intravenous administration of either meclofenamate or indomethacin reduced uterine vein PGE to 23 ng/ml (P less than 0.01) and arterial PGE to 1.0 ng/ml (P less than 0.05). Male and nonpregnant female rabbits had lower arterial PGE, 0.37 ng/ml (P less 0.05). Studies in non-nephrectomized pregnant animals demonstrated that uteroplacental secretion of PGE was greater than five times renal secretion. These studies demonstrate that the rabbit uteroplacental unit is a rich source of PGE and suggest that production of the vasoactive lipid may have a key role in regulating UPBF during pregnancy.
R C Venuto, T O'Dorisio, J H Stein, T F Ferris
The influence of Tuftsin, the synthetic phagocytosis-stimulating tetrapeptide (L-threonyl-L-lysyl-L-prolyl-L-arginine), on the nitrous blue tetrazolium (NBT) reduction by human polymorphonuclear leukocytes was investigated. It was found that this substance increases the NBT reduction by approximately as much as endotoxin. Other tetrapeptides do not share this property. When Tuftsin analogs are added to the cell suspension and incubated, they prevent the action of both Tuftsin and endotoxin but not of methylene blue. When washed of the analogs, the cells regain the property to be activated by both Tuftsin and endotoxin. It appears that methylene blue on one hand and Tuftsin and endotoxin on the other hand have different sites for their actions. We suggest that whereas methylene blue diffuses into the cell and acts directly upon the hexosemonophosphate shunt activation, Tuftsin and endotoxin appear to act on the cell membrane binding to specific receptors. By treating the cells with Tuftsin analogs, we probably block these receptors.
Z Spirer, V Zakuth, A Golander, N Bogair, M Fridkin
To delineate the role of estradiol in the augmented pituitary gonadotropin responsiveness to synthetic luteinizing hormone releasing factor (LRF) seen during high-estrogen phases of the ovulatory cycles (late follicular and midluteal phases), the anti-estrogenic effect of clomiphene citrate (Clomid) on pituitary response to LRF was evaluated during different phases of the ovulatory cycle. Clomid administration (100 mg/day times 5 days) completely negates the augmented gonadotropin responses to LRF (150 mug) during late follicular and midluteal phases observed during the control studies. Thus, a quantitatively and qualitatively similar pituitary sensitivity to LRF during three distinct phases of the menstrual cycle was induced by Clomid treatment that resembles the LRF responsiveness of themale pituitary. The present study demonstrates the pituitary component of the estrogen-induced changes in the sensitivity to LRF. From this and previous data, we conclude that the increases of estradiol secretion associated with the follicular maturation and corpus luteum formation represent a major component of the feedback signal in the modulation of cyclic gonadotropin release occasioned in a large measure by the augmented pituitary sensitivity to LRF.
C F Wang, S S Yen