Issue published October 1, 1977

N ephrogenous cyclic AMP (NcAMP), total cyclic AMP excretion (UcAMP), and plasma immunoreactive parathyroid hormone (iPTH), determined with a multivalent antiserum, were prospectively measured in 55 control subjects, 57 patients with primary hyperparathyroidism (1°HPT), and 10 patients with chronic hypoparathyroidism.In the group with 1° HPT, NcAMP was elevated in 52 patients (91%), and similar elevations were noted in subgroups of 26 patients with mild (serum calcium ≤10.7 mg/dl) or intermittent hypercalcemia, 19 patients with mild renal insufficiency (mean glomerular filtration rate, 64 ml/min), and 10 patients with moderate renal insufficiency (mean glomerular filtration rate, 43 ml/min). Plasma iPTH was increased in 41 patients (73%).The development of a parametric expression for UcAMP was found to be critically important in the clinical interpretation of results for total cAMP excretion. Because of renal impairment in a large number of patients, the absolute excretion rate of cAMP correlated poorly with the hyperparathyroid state. Expressed as a function of creatinine excretion, UcAMP was elevated in 81% of patients with 1° HPT, but the nonparametric nature of the expression led to a number of interpretive difficulties. The expression of cAMP excretion as a function of glomerular filtration rate was developed on the basis of the unique features of cAMP clearance in man, and this expression, which provided elevated values in 51 (89%) of the patients with 1° HPT, avoided entirely the inadequacies of alternative expressions.Results for NcAMP and UcAMP in nonazotemic and azotemic patients with hypoparathyroidism confirmed the validity of the measurements and the expressions employed.

T he serum of some patients with insulin-resistant “diabetes” contains antibodies that bind to and block the cell membrane receptors for insulin. In this report, we have characterized the effects of the antireceptor antibodies on the interaction of ¹²⁵I-insulin with its receptor on the human lymphoblastoid cell line IM-9. Up to 95% of specific insulin binding can be inhibited by pretreatment of the cells with these immunoglobulins. The onset of the inhibitory effect is time- and temperature-dependent, and the effect is reversed extremely slowly if the cells are suspended in a large excess of antibody-free buffer. These features of antibody binding can be easily distinguished from those for insulin binding to its receptor. The inhibitory effect of the antibodies can be reversed by exposure of the cells to conditions known to elute surface immunoglobulins.The three antireceptor sera studied appear to alter the insulin-receptor interaction in different ways. Two antisera markedly reduce receptor affinity through combined effects on the insulin association and dissociation rates, and, additionally, have smaller effects on available receptor number. A third antiserum primarily affects available receptor number and has little effect on receptor affinity. All three antisera inhibit the capacity of insulin to promote negatively cooperative site-site interactions among insulin receptors. The data suggest that these autoantibodies to the insulin receptor bind to different determinants on the receptor and may therefore be useful as unique probes of insulin receptor structure and function.

T he turnover of ¹²⁵I-high density lipoprotein (HDL) was examined in a total of 14 studies in eight normal volunteers in an attempt to determine the metabolic relationship between apolipoproteins A-I (apoA-I) and A-II (apoA-II) of HDL and to define further some of the determinants of HDL metabolism. All subjects were first studied under conditions of an isocaloric balanced diet (40% fat, 40% carbohydrate). Four were then studied with an 80% carbohydrate diet, and two were studied while receiving nicotinic acid (1 g three times daily) and ingesting the same isocaloric balanced diet. The decay of autologous ¹²⁵I-HDL and the appearance of urinary radioactivity were followed for at least 2 wk in each study. ApoA-I and apoA-II were isolated by Sephadex G-200 chromatography from serial plasma samples in each study. The specific activities of these peptides were then measured directly.It was found that the decay of specific activity of apoA-I and apoA-II were parallel to one another in all studies. The mean half-life of the terminal portion of decay was 5.8 days during the studies with a balanced diet.Mathematical modeling of the decay of plasma radioactivity and appearance of urinary radioactivity was most consistent with a two-compartment model. One compartment is within the plasma and exchanges with a nonplasma component. Catabolism occurs from both of these compartments.With a balanced isocaloric diet, the mean synthetic rate for HDL protein was 8.51 mg/kg per day. HDL synthesis was not altered by the high carbohydrate diet and was only slightly decreased by nicotinic acid treatment. These perturbations had effects on HDL catabolic pathways that were reciprocal in many respects. With an 80% carbohydrate diet, the rate of catabolism from the plasma compartment rose by a mean of 39.1%; with nicotinic acid treatment, it fell by 42.2%. Changes in the rate of catabolism from the second compartment were generally opposite those in the rate of catabolism from the plasma compartment, suggesting that these two catabolic pathways may be reciprocally regulated.

T he mechanisms of uptake of parathyroid hormone (PTH) by the kidney was studied in anesthetized dogs before and after ureteral ligation. During constant infusion of bovine PTH (b-PTH 1-84), the renal arteriovenous (A-V) difference for immunoreactive PTH (i-PTH) was 22±2%. After ureteral ligation and no change in renal plasma flow, A-V i-PTH fell to 15±1% (P < 0.01), indicating continued and significant uptake of i-PTH at peritubular sites and a lesser role of glomerular filtration (GF) in the renal uptake of i-PTH. Since, under normal conditions, minimal i-PTH appears in the final urine, the contribution of GF and subsequent tubular reabsorption was further examined in isolated perfused dog kidneys before and after inhibition of tubular reabsorption by potassium cyanide. Urinary i-PTH per 100 ml GF rose from 8±4 ng/min (control) to 170±45 ng/min after potassium cyanide. Thus, i-PTH is normally filtered and reabsorbed by the tubular cells. The physiological role of these two mechanisms of renal PTH uptake was examined by giving single injections of b-PTH 1-84 or synthetic b-PTH 1-34 in the presence of established ureteral ligation. After injection of b-PTH 1-84, renal A-V i-PTH was 20% only while biologically active intact PTH was present (15-20 min). No peritubular uptake of carboxyl terminal PTH fragments was demonstrable. In contrast, after injection of synthetic b-PTH 1-34, renal extraction of N-terminal i-PTH after ureteral ligation (which was 13.4±0.6% vs. 19.6±0.9% in controls) continued for as long as i-PTH persisted in the circulation. These studies indicate that both GF and peritubular uptake are important mechanisms for renal PTH uptake. Renal uptake of carboxyl terminal fragments of PTH is dependent exclusively upon GF and tubular reabsorption, whereas peritubular uptake can only be demonstrated for biologically active b-PTH 1-84 and synthetic b-PTH 1-34.

T he effects of estrogen and progesterone on uterine alpha-adrenergic receptors were investigated by direct receptor-binding studies. Immature female rabbits were primed with estrogen by intramuscular injections for 4 days. Other rabbits were primed with progesterone by injections of estrogen for 4 days followed by injections of progesterone for 4 days. The alpha adrenergic antagonist, [3H]dihydroergocryptine, was used to directly assess the number and affinity of alpha adrenergic receptors in membranes derived from estrogen-and progesterone-primed uteri. Membranes from estrogen-primed uteri contained 257 +/- 52 fmol of [3H]dihydroergocryptine-binding sites per mg protein whereas membranes from progesterone-primed uteri contained 83 +/- 11 fmol of of binding sites per mg protein. This reduction of alpha adrenergic receptor-binding sites by progesterone was statistically significant (P less than 0.02). In contrast, no significant difference in the binding site affinity was observed between the estrogen- and progesterone-primed groups. The progesterone-induced decrease in the number of uterine alpha adrenergic receptors provides a potential explanation for the reduced alpha adrenergic contractile response to epinephrine in the progesterone-primed myometrium.

W hen Factor VIII/von Willebrand factor (FVIII/vWF) protein is rechromatographed on 4% agarose in 0.25 M CaCl₂, the protein and vWF activity appear in the void volume, but most of the FVIII procoagulant activity elutes later. Recent evidence suggests that the delayed FVIII procoagulant activity is a proteolytically modified form of FVIII/vWF protein that filters anomalously from agarose in 0.25 M CaCl₂. To test whether or not thrombin is the protease involved, the effect of 0.25 M CaCl₂ on FVIII/vWF and its reaction with thrombin was examined. About 30% of the FVIII procoagulant activity was lost immediately when solutions of FVIII/vWF protein were made 0.25 M in CaCl₂. When FVIII in 0.15 M NaCl was activated with 0.04 U thrombin/ml and then made 0.25 M in CaCl₂, the procoagulant activity of a broad range of FVIII/vWF protein concentrations remained activated for at least 6 h. But, in 0.25 M CaCl₂, the increase in FVIII procoagulant activity in response to thrombin was much more gradual and once activated, the procoagulant activity was stabilized by 0.25 M CaCl₂. When thrombin-activated FVIII/vWF protein was filtered on 4% agarose in 0.15 M NaCl, there was considerable inactivation of FVIII procoagulant activity; however, the procoagulant activity that did remain eluted in the void volume. In contrast, when thrombin-activated FVIII/vWF protein was filtered in 0.25 M CaCl₂, the FVIII procoagulant activity eluted well after the void volume and remained activated for 6 h. The procoagulant peak isolated by filtering nonthrombin-activated FVIII/vWF protein on agarose in 0.25 M CaCl₂ was compared to that isolated from thrombin-activated FVIII/vWF protein. Both procoagulant activity peak proteins had about the same specific vWF activity as the corresponding void volume protein. Before reduction, the sodium dodecyl sulfate gel patterns for the two procoagulant activity peak proteins were the same. After reduction, the gel pattern for the nonthrombin-activated procoagulant activity peak protein contained bands of 195,000, 148,000-120,000, 79,000, 61,000, 51,000, and 18,000 daltons whereas the pattern for the reduced thrombin-activated procoagulant activity peak protein always lacked the higher molecular weight bands, but consistently showed the four lower molecular weight bands to be well resolved. Taken together, these results imply that thrombin generates the FVIII procoagulant activity that is stabilized by 0.25 M CaCl₂ and elutes aberrantly from 4% agarose in that solvent.

S tandard clearance studies were performed in mechanically ventilated intact and acutely thyroparathyroidectomized (TPTX) rats to document and characterize the effect of hypercapnia (HC) on urinary phosphorus excretion (U_PV). HC as compared to normocapnia (NC) was associated with an increase in U_PV in intact (62.5 vs. 7.93 μg/min) and TPTX (30.5 vs. 0.59 μg/min) rats, an increase in filtered load of phosphorus in intact (218 vs. 191 μg/min) and TPTX (243 vs. 146 μg/min) rats, an increase in blood bicarbonate concentration in intact (27.8 vs. 26.0 meq/liter) and TPTX (24.5 vs. 22.3 meq/liter) animals, and a decrease in blood pH in intact (7.15 vs. 7.42) and TPTX (7.07 vs. 7.39) rats. Additional TPTX rats with NC and HC were studied during phosphorus infusion at a comparable filtered load of phosphorus (NC = 307 μg/min and HC = 328 μg/min). U_PV was 18.5 μg/min in NC and 85.2 μg/min in HC animals. Intact NC animals infused with NaHCO₃ achieved a blood bicarbonate of 45.9 meq/liter compared to 26.0 meq/liter in intact NC NaCl-infused rats. U_PV was 10.0 μg/min in the NaHCO₃ and 7.93 μg/min in NaCl-infused animals. In intact HC animals infused with NaHCO₃, blood pH was 7.36 compared to 7.42 in NC intact NaCl-infused animals. U_PV was 83.2 μg/min in the HC bicarbonate-infused and 7.93 μg/min in the NC NaCl-infused rats. These experiments demonstrate that elevated blood carbon dioxide tension per se increases U_PV. Increases in filtered load of phosphorus and blood bicarbonate which are associated with HC contribute to the phosphaturia as does parathyroid hormone. The phosphaturia is not dependent upon reduction of extracellular pH.

T he occurrence of circulating immune complexes was investigated in 31 patients with cytomegalovirus infection (29 infected in utero and 2 with natal infection) and 34 uninfected controls. Anti-complementary activity above 1:20 occurred in 34% (29/86) of the sera tested from the infected group in contrast to 7.5% (3/40) in the controls (P < 0.005). When assayed by means of a lymphoblastoid cell line (Raji cell test), the reactivity in these groups was 45 (39/86) and 2.7% (1/36), respectively (P < 0.001). Correlation of results between these two complement-dependent assays occurred in 75% of samples collected from the infected group. Frequency of reactivity was higher in severe intrauterine infection and during the 1st yr of life paralleling the patterns of viral excretion and humoral immune responses. Physicochemical characterization demonstrated that reactive substances in sera were acid-dissociable and, in one sample tested, contained 7S IgG antibodies with cytomegalovirus (CMV) specificity. Circulating immune complexes were heavier (18-22S) in sick, as opposed to subclinically CMV-infected patients, in whom intermediate size complexes (12-16S) were found. In three of four symptomatic patients whose demise was due to severe congenital infection, granular deposits of immunoglobulins and C3 were detected in a pattern typical of immune complexes along the glomerular basal membrane of the glomeruli. Whether or not circulation and deposition of heavier immune complexes contributed to the adverse clinical outcome is unresolved. Because of the high incidence of both congenital and natal CMV infections, definition of the pathogenetic potentials of both heavy and intermediate size immune complexes is required to design more effective therapeutic measures.

T he ability to clone primary tumors in soft agar has proven useful in the study of the kinetics and biological properties of tumor stem cells. We report the development of an in vitro assay which permits formation of colonies of human monoclonal plasma cells in soft agar. Colony growth has been observed from bone marrow aspirates from 75% of the 70 patients with multiple myeloma or related monoclonal disorders studied. Growth was induced with either 0.02 ml of human type O erythrocytes or 0.25 ml of medium conditioned by the adherent spleen cells of mineral oil-primed BALB/c mice. 5-500 colonies appeared after 2-3 wk in culture yielding a plating efficiency of 0.001-0.1%. The number of myeloma colonies was proportional to the number of cells plated between concentrations of 10⁵-10⁶ and back-extrapolated through zero, suggesting that colonies were clones derived from single myeloma stem cells. Morphological, histochemical, and functional criteria showed the colonies to consist of immature plasmablasts and mature plasma cells. 60-80% of cells picked from colonies contained intracytoplasmic monoclonal immunoglobulin. Colony growth was most easily achieved from the bone marrow cells of untreated patients or those in relapse. Only 50% of bone marrow samples from patients in remission were successfully cultured. Tritiated thymidine suicide studies provided evidence that for most myeloma patients, a very high proportion of myeloma colony-forming cells was actively in transit through the cell cycle. Velocity sedimentation at 1 g showed myeloma stem cells sedimented in a broad band with a peak at 13 mm/h. Antibody to granulocyte colony-stimulating factor did not reduce the number or size of the colonies. Increased numbers of myeloma colonies were seen when the marrow was depleted of colony-stimulating factor elaborating adherent cells before plating. This bioassay should prove useful in studying the in vitro biological behavior of certain bone marrow-derived (B)-cell neoplasia. In addition, systematic and predictive studies of anticancer drug effects on myeloma stem cells should now be feasible.

F ibrinogen and the cold-insoluble globulin of plasma (CIg) are the main protein components of the heparin-precipitable fraction of normal plasma. The interactions among these proteins and heparin were examined. Heparin formed a cold-precipitable complex with purified CIg or with mixtures of CIg and fibrinogen but not with purified fibrinogen alone. Cryoprecipitation was augmented by addition of Ca⁺⁺ or by selection of optimal heparin levels; it was reduced or even abolished by raising the ionic strength or pH or both, or by raising the heparin concentration above that for maximum precipitation of CIg. Fibrinogen reduced the threshold for heparin-induced CIg cryoprecipitation and, by coprecipitating with heparin and CIg, increased the amount of precipitate that formed. In contrast to the heparin-precipitable fraction of normal plasma which contained both fibrinogen and CIg, that from a patient with congenital afibrinogenemia contained CIg but lacked fibrinogen. Normal plasma depleted of CIg by immunoabsorption failed to form a heparin-induced cryoprecipitate. Thus, CIg is essential for heparin-induced cryoprecipitation to occur. Fibrinogen, as assessed by chromatographic experiments with heparin-Sepharose columns, had a considerably lower binding affinity for heparin than did CIg, suggesting that it participates in precipitate formation mainly, if not entirely, by virtue of its affinity for CIg. The region of the fibrinogen molecule accounting for its precipitation with CIg appears to be localized in the carboxy-terminal segment of the Aα-chain; fibrinogen subfractions lacking this region failed to augment cryoprecipitation of heparin-CIg mixtures and, even though such species were present in normal plasma, they failed to coprecipitate in the heparin-induced complex.

T he mechanism of stimulus-response coupling in human platelets was investigated with a new instrument that simultaneously monitors aggregation and secretion in the same sample of plateletrich plasma. When platelets were stimulated by high concentrations of ADP, secretion began only after aggregation was almost complete. With lower concentrations of ADP or with epinephrine, biphasic aggregation was observed, and secretion began simultaneously with, or slightly after, the second phase of aggregation. When platelets were stimulated with high concentrations of γ-thrombin or A23187, secretion and aggregation began essentially together. With very low concentrations of γ-thrombin or A23187, biphasic aggregation was observed with secretion paralleling the second phase. At every concentration of collagen, secretion and aggregation appeared to be parallel events. Under every condition where the beginning of secretion lagged behind aggregation, secretion was dependent upon aggregation and was inhibited by indomethacin; this is referred to as aggregation-mediated platelet activation. When secretion began at the same time as aggregation, it also occurred in the absence of aggregation and was not blocked by indomethacin; this is referred to as directly induced platelet activation. These observations are consistent with a simple model of platelet stimulus-response coupling that includes two mechanisms for activation; aggregation-mediated activation is inhibited by indomethacin, while direct activation does not depend upon aggregation and is not inhibited by indomethacin. Secretion and second wave aggregation appear to be parallel events, with little evidence for second wave aggregation being a consequence of secretion as usually described.

T he occurrence of circulating immune complexes was investigated in 467 serum samples from 230 leukemia patients using the [¹²⁵I]Clq-binding test. There was an increased serum [¹²⁵I]Clq-binding activity in 40% of patients with acute myeloid leukemia, 23% with acute lymphatic leukemia, 46% in blastic crisis of chronic myeloid leukemia, 12% with chronic lymphatic leukemia, and 13% with chronic myeloid leukemia. In 48 patients, serum was also tested for soluble immune complexes by the Raji cell radioassay; the correlation between results of the two tests was significant. The Clq-binding material had properties identical with those of immune complexes. It sedimented as 14-28s material on sucrose density gradient. It contained IgG which could be dissociated at acid pH. Its Clq-binding properties could be removed after passage through anti-IgG immuno-absorbant or after a mild reduction-alkylation treatment, but were not sensitive to deoxyribonuclease treatment. Circulating immune complexes were found most commonly during the blastic stage of leukemia.Remission took place in 75.4% of patients with no detectable circulating immune complexes at the onset of acute leukemia, but in only 32.7% of those with detected complexes during this period. Median survival times of the former group of patients were more than 18 mo in acute myeloid leukemia and acute lymphatic leukemia and more than 8½ mo in blastic crisis of chronic myeloid leukemia. The corresponding median survival times in the latter patient group were 64, 135, and 90 days. These findings were unrelated to prognostic features already known.

R ecent epidemiologic studies have suggested that cardiac disease in common in diabetics and may often have a noncoronary basis. To examine the status of the left ventricle, 17 adult-onset diabetics of familial type without hypertension or obesity underwent hemodynamic study and were compared to 9 controls of similar age.Of the 17, 12 subjects had no significant occlusive lesions by coronary angiography. From this group eight without heart failure had a modest, but significant, elevation of left ventricular end-diastolic pressure. End-diastolic and stroke volumes were reduced, but ejection fraction and mean rate of fiber shortening were within normal limits. The left ventricular end-diastolic pressure/volume ratio was significantly higher than controls. Afterload increments effected a significant increase of filling pressure compared to normals without a stroke volume response, consistent with a preclinical cardiomyopathy. Four patients with prior heart failure had similar but more extensive abnormalities. None had local dyskinesia by angiography, and lactate production was not observed during pacing-induced tachycardia. Left ventricular biopsy in two patients without ventricular decompensation showed interstitial collagen deposition with relatively normal muscle cells. These findings suggest a myopathic process without ischemia.Postmortem studies were performed in 11 uncomplicated diabetics. Nine were without significant obstructive disease of the proximal coronary arteries, and the majority succumbed with cardiac failure. On left ventricular sections, none had evident luminal narrowing of the intramural vessels. All nine exhibited periodic acid-Schiff-positive material in the interstitium. Collagen accumulation was present in perivascular loci, between myofibers, or as replacement fibrosis. Multiple samples of left ventricle and septum revealed enhanced triglyceride and cholesterol concentrations, as compared to controls. Thus, a diffuse extravascular abnormality may be a basis for cardiomyopathic features in diabetes.

M uscular exercise is associated with hypermetabolism and increased hypoxic ventilatory response (HVR). In order to dissociate mechanical and metabolic factors, the effect of hypermetabolism on hypoxic ventilatory response was evaluated at rest. Carbohydrate and protein feeding increases metabolic rate, and their effects on chemosensitivity, ventilation, and blood pH were evaluated in six normal subjects 2 h and 3 h after calorically equal test meals (1,000 cal).After carbohydrate, base-line oxygen consumption (V̇o₂) increased from 237±11.3 ml/min (SEM) to 302±19.4 (P < 0.001) and 303±18.5 (P < 0.001) at 2 h and 3 h, respectively. Hypoxic ventilatory response, measured as shape parameter A, increased from a control of 144±11.8 to 330±61.0 (P < 0.01) at 2 h and 286±57.0 (P < 0.05) at 3 h. These changes were associated with a mild metabolic acidosis as pH decreased from a control of 7.402±0.004 to 7.371±0.009 (P < 0.005) at 2 h and 7.377±0.008 (P < 0.005) at 3 h.After protein, V̇o₂ increased from 241±6.7 to 265±6.2 (P < 0.02) and 270±5.4 (P < 0.001), an overall increase less than that which occurred after carbohydrate (P < 0.01). Hypoxic ventilatory response increased from 105±14.5 to 198±24.3 (P < 0.02) at 2 h and 219±17.3 (P < 0.01) at 3 h, which was not different from the increase with carbohydrate. After protein, no acidosis occurred. Thus, after protein, HVR increased despite the absence of a systemic acidosis.We conclude that both carbohydrate and protein feedings are associated with resting hypermetabolism and increased HVR compared with the fasting state. For both meals, increased metabolic rate was correlated with increased hypoxic response.

T he erythropoietic effect of various thyroid hormones has been studied using erythroid colony formation by canine marrow cells. Although erythropoietin was required for colony growth, physiologic levels of thyroid hormones significantly enhanced colony numbers. The order of potency of the thyroid compounds in their in vitro erythropoietic effect parallels their known calorigenic potency in vivo, suggesting that the in vitro effect is physiologically relevant. A series of studies linked the mechanism of thyroid action to adrenergic receptors on responsive cells. Propranolol, a global β-blocker, inhibited thyroid hormone-responsive erythroid colonies. When adrenergic antagonists having different blocking characteristics were added to culture, the thyroid hormone effect was blocked by those compounds having β₂-subspecificity. Velocity sedimentation analysis showed that the peak of colony-forming cells which respond to thyroid hormone and the adrenergic agonist, isoproterenol, sedimented at an identical rate (7.54 mm/h), which is slower than the major peak of colony-forming cells responding to erythropoietin alone (8.62 mm/h). These results demonstrate thyroid hormonal enhancement of in vitro erythroid colony growth which appears mediated by a receptor with β₂-adrenergic properties. The data suggest that changes in hormone-target cell interaction may occur during states of abnormal thyroid function.

I mmunoprecipitates containing guinea pig Factor VIII antigen were prepared from guinea pig plasma with a cross-reacting rabbit anti-human Factor VIII. Monospecific antisera to guinea pig Factor VIII antigen were produced in rabbits by using these washed immunoprecipitates as immunogens. The resulting antisera to guinea pig Factor VIII antigen detected Factor VIII antigen in guinea pig plasma and inhibited the von Willebrand factor activity in guinea pig plasma. This antibody also detected Factor VIII antigen in a solubilized protein mixture prepared from isolated cultured guinea pig megakaryocytes. Cultured guinea pig megakaryocytes were labeled with radio-active leucine. By radioautography, 96.2% of the radio-activity was present in megakaryocytes. The radio-active Factor VIII antigen present in the solubilized cell protein mixture was isolated by immunoprecipitation and characterized by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The results demonstrate that cultured guinea pig megakaryocytes synthesize Factor VIII antigen which contains the same polypeptide subunit (mol wt 200,000) present in guinea pig plasma Factor VIII antigen.

M easurement of urine to blood (U-B) carbon dioxide tension (P_CO2) gradient during alkalinization of the urine has been suggested to assess distal H⁺ secretion. A fact that has not been considered in previous studies dealing with urinary P_CO2 is that dissolution of HCO₃ in water results in elevation of P_CO2 which is directly proportional to the HCO₃ concentration. To investigate the interrelationship of urinary HCO₃ and urinary acidification, we measured U-B P_CO2 in (a) the presence of enhanced H⁺ secretion and decreased concentrating ability i.e., chronic renal failure (CRF), (b) animals with normal H⁺ secretion and decreased concentrating ability, Brattleboro (BB) rats, and (c) the presence of both impaired H⁺ secretion and concentrating ability (LiCl treatment and after release of unilateral ureteral obstruction). At moderately elevated plasma HCO₃ levels (30-40 meq/liter), normal rats achieved a highly alkaline urine (urine pH > 7.8) and raised urine HCO₃ concentration and U-B P_CO2. At similar plasma HCO₃ levels, BB rats had a much higher fractional water excretion and failed to raise urine pH, urine HCO₃ concentration, and U-B P_CO2 normally. At a very high plasma HCO₃ (>50 meq/liter), BB rats raised urine pH, urine HCO₃ concentration, and U-B P_CO2 to the same levels seen in normals. CRF rats failed to raise urine pH, urine HCO₃, and U-B P_CO2 normally at moderately elevated plasma HCO₃ levels; at very high plasma HCO₃ levels, CRF rats achieved a highly alkaline urine but failed to raise U-B P_CO2. Dogs and patients with CRF were also unable to raise urine pH, urine HCO₃ concentration, and U-B P_CO2 normally at moderately elevated plasma HCO₃ levels. In rats, dogs, and man, U-B P_CO2 was directly related to urine HCO₃ concentration and inversely related to fractional water excretion. At moderately elevated plasma HCO₃ levels, animals with a distal acidification defect failed to raise U-B P_CO2; increasing the plasma HCO₃ to very high levels resulted in a significant increase in urine HCO₃ concentration and U-B P_CO2. The observed urinary P_CO2 was very close to the P_CO2 which would be expected by simple dissolution of a comparable amount of HCO₃ in water. These data demonstrate that, in highly alkaline urine, urinary P_CO2 is largely determined by concentration of urinary HCO₃ and cannot be used as solely indicating distal H⁺ secretion.

T he interactions of dipyridamole with α₁ acid glycoprotein of plasma and with human platelets are related to inhibition of adenosine uptake by platelets. Binding studies by equilibrium gel filtration suggested that 1 mol of dipyridamole binds per mol of α₁ acid glycoprotein with a dissociation constant of 1.6 μM. Platelets contain two populations of binding sites, one with high and another with lower affinity for the drug. The binding of dipyridamole to the high-affinity sites follows a Michaelis-Menten binding pattern with a dissociation constant of 0.04 μM. Approximately 2 × 10⁴ dipyridamole molecules are bound at the high-affinity sites of each platelet. The lower affinity sites bind the drug with a dissociation constant of 4 μM. In the presence of α₁ acid glycoprotein of plasma, the binding of dipyridamole to human platelets is inhibited. Correspondingly, the dipyridamole inhibition of adenosine uptake by platelets is reduced 1,000-fold by purified α₁ acid glycoprotein. The binding of dipyridamole to human platelets was found to be essential for its inhibition of adenosine uptake by platelets. Dipyridamole decreases the incorporation of [¹⁴C]adenosine radioactivity in platelet nucleotides and reduces the [¹⁴C]-ATP to [¹⁴C]ADP ratio. Purified α₁ acid glycoprotein reverses these effects of dipyridamole on adenosine metabolism of platelets in a concentration-dependent manner. An equilibrium of dipyridamole binding to α₁ acid glycoprotein and to platelets is proposed.

A n arginine-rich apoprotein obtained from human triglyceride-rich lipoprotein was isolated on a heparin affinity column when either the aqueousor urea-soluble apoproteins were applied to the column. Of all the aqueous- or urea-soluble apoproteins, only this arginine-rich protein exhibited a binding affinity to heparin. This protein was eluted from the column at sodium chloride concentrations above 0.35 M in the absence of urea and between 0.17-0.2 M when isolated in urea. The protein has been characterized by amino acid analysis, immunoelectrophoresis, dodecyl sulfate polyacrylamide electrophoresis, isoelectric focusing, and NH₂-terminal analysis. It has the same amino acid composition, NH₂-terminal, and molecular weight as previously described for human arginine-rich apoprotein.The triglyceride-rich lipoproteins of fasting normal humans were eluted as two fractions when applied to the heparin affinity column. A small amount was eluted in the unbound fraction and this species contained virtually no arginine-rich apoprotein. The bulk of the triglyceride-rich lipoproteins eluted in the bound fraction and contained appreciable amounts of arginine-rich apoprotein. The bound lipoproteins had more cholesterol and cholesterol ester and less triglyceride than the unbound. The isolated arginine-rich apoprotein was derivatized with phenylglyoxal with a resulting alteration of 75% of the arginine residues. This modified apoprotein did not bind to the heparin affinity column. Similar treatment of the whole triglyceride-rich lipoprotein produced a lipoprotein that was totally eluted in the unbound fraction.

T he addition of specific antigen to IgE-sensitized human lung tissue causes the secretion of the mediators histamine and slow-reacting substance of anaphylaxis. The mechanisms by which increased levels of cyclic AMP suppress and increased levels of cyclic GMP enhance this secretory process were studied. Colchicine, an agent which disrupts many secretory reactions by binding to microtubules in their disassembled 6S form, was a relatively ineffective inhibitor of the antigen-induced release of mediators unless lung fragments were incubated at 4°C for 60 min to induce microtubular disassembly. As colchicine appeared to inhibit the immunologic secretion of mediators from human lung tissue most effectively after microtubular disassembly, the capacity of colchicine to modulate the release reaction indicated the state of microtubular assembly; inhibition by colchicine signaled a shift to the colchicine-sensitive 6S subunits whereas failure to inhibit suggested maintenance in the colchicine-resistant polymerized state.Exogenously added 8-Bromo-cyclic GMP prevented low temperature-facilitated colchicine suppression of mediator release suggesting that increased levels of cyclic GMP stabilize polymerized microtubules. Transiently increased cyclic AMP concentrations, either exogenously added as 8-Bromo-cyclic AMP or endogenously produced by isoproterenol, promoted colchicine suppression of mediator release suggesting that microtubular disassembly was produced. Direct measurement of cyclic AMP levels revealed parallel kinetics after isoproterenol stimulation between control and colchicine-treated lung fragments.The requirement for functional microtubules in the release reaction may occur after the antigen IgE-stimulated activation of a serine esterase, energy utilization, and an intracellular calcium requirement. The mechanism by which cyclic nucleotides influence microtubular assembly is postulated to involve the degree of phosphorylation-dephosphorylation of microtubules.