Cigarette smoke increases gastric ulcer size in part by an angiotensin II-mediated mechanism in rats.

To assess the mechanism of the effect of cigarette smoke on ulcer disease we employed a rat model in which cigarette smoke increases the size of acetic acid-induced gastric ulcer and decreases the hyperemia at the ulcer margin. We postulate that cigarette smoke increases angiotensin II (a vasoconstrictor) in ulcer tissue. Since direct measurement of angiotensin II in small tissue samples is problematic, we compared the messenger ribonucleic acid (mRNA) for its precursors (angiotensinogen and renin) in ulcer and normal gastric tissue. We also evaluated the effect of enalapril, which blocks the conversion of angiotensin I to angiotensin II on ulcer size. In the ulcer tissue, cigarette smoke produced a significant increase in mRNA for angiotensinogen but not for renin. Enalapril decreased the size of the gastric ulcer in rats exposed to cigarette smoke. The data support the possibility that in ulcer tissue cigarette smoke stimulates an angiotensin II-mediated mechanism, which may in part be responsible for the impairment of ulcer margin hyperemia and aggravation of ulcer size.

Hepatic angiotensin II nuclear receptors and transcription of growth-related factors.

OBJECTIVES: To investigate the influence of angiotensin II (All) receptors in isolated hepatic nuclei on other genes regulated by All and to determine whether the function of these intracellular receptors is influenced by alterations in the endocrine renin system. METHODS: Nuclei were isolated from hepatic tissue of normal and bilaterally nephrectomized or adrenalectomized Wistar rats. Following nuclear run-off, in the presence of varying All concentrations, specific messenger RNAs (mRNA) were determined by slot blot hybridization. Tissue levels of renin system components were measured by radioimmunoassay and nuclear receptors characterized by displacement of radiolabeled All with specific All receptor antagonists. RESULTS: All binding in the presence of DUP 753 and PD 123177 confirmed that nuclear All receptors can be classified as AT1 receptors and that as much as 10% of the specific binding is attributable to nuclear chromatin. All stimulated not only the production of mRNA for renin system components such as renin and angiotensinogen, but also that of mRNA for growth-related factors such as platelet-derived growth factor and the oncogene c-myc. Maximal stimulation occurred at 10(-9) mol/l All; higher concentrations reduced this response. After stimulation or suppression of the plasma renin system by adrenalectomy or bilateral nephrectomy, nuclei isolated from rat hepatic tissue contained elevated endogenous levels of growth-related and renin system mRNA including AT1 and AT2 All receptors. However, despite the level of receptor mRNA having been elevated, the total All receptor density of isolated nuclei decreased. In addition, after both maneuvers, isolated nuclei were refractory to All-induced gene transcription. CONCLUSION: The existence of mechanisms producing intracellular All and regulating its level, which in turn exert local regulatory responses via nuclear All receptors, lends significance to the presence of a functional intracrine renin system that could act in concert with or independently of the endocrine renin system.

Cloning and functional characterization of the amphibian mesotocin receptor, a member of the oxytocin/vasopressin receptor superfamily.

Mesotocin is the oxytocin-like hormone found in most terrestrial vertebrates from lungfishes to marsupials, which includes all non-mammalian tetrapods (amphibians, reptiles, and birds). It has the largest distribution in vertebrates after vasotocin found in all non-mammalian vertebrates and isotocin identified in bony fishes. In this study, we report the cloning and functional characterization of the cDNA for the mesotocin receptor (MTR) from the urinary bladder of the toad Bufo marinus. The cloned cDNA encodes a polypeptide of 389 amino acids that shows the greatest similarity to the teleost fish isotocin receptor and to mammalian oxytocin receptors with mutations in extracellular loops which are involved in ligand binding. When expressed in COSM6 cells, MTR exhibits the following relative order of ligand affinity: mesotocin > vasotocin = oxytocin > vasopressin > hydrin 1, isotocin, hydrin 2. Injection of MTR cRNA into Xenopus laevis oocytes induces membrane chloride currents in response to mesotocin, which indicates the coupling of the mesotocin receptor to the inositol phosphate/calcium pathway. This response is inhibited by an oxytocin antagonist, but not by a vasopressin antagonist specific for V2 vasopressin receptors. MTR mRNA is not only found in toad urinary bladder, but also in kidney, muscle, and brain tissue of the toad as revealed by northern blot analysis and reverse-transcriptase PCR. The results suggest a variety of function for mesotocin and its receptor including, in particular, an involvement in the regulation of water and salt transport.

Long-term effects of transdermal estradiol with and without medroxyprogesterone acetate.

OBJECTIVE: To study the long-term biological and metabolical effects of estradiol (E2) administered by transdermal therapeutic systems with and without the addition of medroxyprogesterone acetate (MPA). DESIGN: Open, randomized, comparative trial. SETTING: The reproductive endocrine unit of a tertiary care university-affiliated hospital. PATIENTS: Fifty-seven postmenopausal women were given E2 transdermally, whereas 28 were randomized to take MPA by mouth. Fifteen premenopausal women were studied for comparison. INTERVENTIONS: Estradiol, 0.1 mg, was administered by a transdermal therapeutic system for 24.5 of 28 days and was cycled for 96 weeks. Medroxyprogesterone acetate, 10 mg, was given for days 13 to 25 of each 28-day cycle (E+P group), whereas the remainder received E2 only. MAIN OUTCOME MEASURES: Serum E2, estrone (E1), luteinizing hormone, follicle-stimulating hormone, low-density, high-density, very low-density, and total cholesterol, triglycerides, blood pressure, renin substrate, plasma renin activity, and serum aldosterone levels were measured in all subjects at baseline and in the postmenopausal women every 24 weeks until the end of study. RESULTS: Mean +/- SE levels of E2 rose significantly from baseline at 24 weeks to 426 and 355 pmol/L for the E only and E+P groups, respectively. Smaller increases of estrone (E1) were observed to 263 and 244 pmol/L for the same respective groups. As expected, baseline levels of both gonadotropins were elevated, fell significantly with E2 administration, but remained increased in comparison with values observed in younger women. Decreases of total and low-density lipoprotein (LDL) cholesterol were observed in both groups that reached statistical significance at 48 weeks or later with the exception of LDL cholesterol in the E only group. No significant change of high-density lipoprotein or very low-density lipoprotein cholesterol or triglycerides was observed. There were reductions of mean systolic and diastolic blood pressures in both groups that reached significance at 72 weeks. Mean baseline plasma renin substrate, plasma renin activity, and serum aldosterone levels were within the ranges observed in younger, healthy women and did not change significantly with E2 administration in either group. CONCLUSION: These data support the long-term efficacy and safety of this form of replacement therapy, particularly in combination with MPA, in women with a uterus.

Photoaffinity, biotinyl, and iodo analogues as probes for vasotocin receptors.

Vasotocin (AVT) analogues with tyrosine or phenylalanine in position 9, i.e., [9-tyrosine]AVT, [2-phenylalanine,-9-tyrosine]AVT, and 1-desamino[9-(p-aminophenylalanine)]AVT, were synthesized by the solid-phase method. These compounds showed a high biological activity in the hydroosmotic toad bladder assay. Using the chemically reactive functional groups on tyrosine and p-aminophenylalanine in position 9, we prepared iodinated, photoreactive, and affinity ligands, i.e., [2-phenylalanine,9-(iodotyrosine)]AVT, 1-desamino[9-(p-azidophenylalanine)]AVT, and 1-desamino[9-(biotinylphenylalanine)]AVT, respectively. Half-maximal hydroosmotic responses (ED-50 values) were obtained with 2.5 X 10(-9) M for the iodinated analogue, with 0.9 X 10(-10) M for the photoaffinity analogue, and with 1.2 X 10(-8) M for the biotinyl analogue. The hydroosmotic activity of the biotinyl analogue was reversed following addition of avidin, whereas the photoaffinity analogue induced a persistent response following UV irradiation that was not reversed upon repeated and prolonged periods of washout. These analogues of vasotocin are the most potent that have been synthesized to date, and they should serve as useful probes in the isolation and characterization of vasotocin receptors in toad bladders and tissues from other species that use vasotocin as an antidiuretic/pressor principle. The photoaffinity and biotinyl analogues had a rat antidiuretic activity of 66 and 40 units/mg, respectively. These compounds are, therefore, also suitable for the isolation of V-2 vasopressin receptors from mammalian tissues.

Effects of trypsin on the measurement of inactive renin in rat plasma by radio-immunoassay: decrease in inactive renin following nephrectomy.

We have examined the effect of trypsin treatment of rat plasma on the rate of angiotensin (Ang) I generation and measurement of this peptide by radio-immunoassay. Trypsin increased the renin incubation blank but did not alter the kinetics of the renin reaction with exogenous renin. The quantity of immunoreactive material detected in trypsin-treated plasma was not proportional to the volume of plasma assayed. Consequently, the level of inactive renin was dependent upon the volume of plasma subjected to the assay. This discrepancy occurred with two independent radio-immunoassay systems. The rate of Ang I generation was linear and significantly elevated following the addition of renin substrate to trypsin-treated plasma. However, if trypsin degradation of endogenous renin substrate was extensive and additional renin substrate was not provided, non-linear rates of Ang I generation occurred. Multiple additions of trypsin were necessary to activate maximally inactive rat plasma renin. Inactive renin accounted for 79 +/- 2% of the total enzyme activity in normal rats. Although active renin declined following bilateral nephrectomy, the ratio of active to inactive renin did not change. The data suggest that the kidney is the primary source of inactive renin in the normal rat.

Stimulation of brain and aortic renin substrate by central administration of steroids in the rat.

The influence of central and peripheral injections of dexamethasone and 17 beta-oestradiol on plasma, brain and aortic renin substrate was studied in the rat. Whereas intraperitoneal or intravenous injection of these steroids raised renin substrate in plasma, brain and the aorta, intraventricular injection raised it only in the brain and aorta. Bilateral nephrectomy produced results equivalent to those observed following intraperitoneal administration of steroids. Dissociation of the plasma and tissue renin-substrate levels in response to central administration of steroids indicates local synthesis of this protein, possibly under central control.

Monoclonal antibodies to pig angiotensinogen recognize minor idiotypes.

We have produced monoclonal antibodies to a highly purified pig (P) angiotensinogen preparation and characterized their ability to bind [125]I-P- angiotensinogen. Lymphocytes of RBF/Dn mice immunized with P-angiotensinogen were fused with FOX-NY myeloma cells and clones were isolated by binding to [125]I-P-angiotensinogen and by an immunodot blot assay. Three of 16 clones which recognized P-angiotensinogen were characterized. Isolated monoclonal antibodies bound only 10-15% of the total [125]I-P-angiotensinogen; however, the bound counts could be displaced with unlabelled P-angiotensinogen. None of the monoclonals inhibited the cleavage of P-angiotensinogen by homologous renin, nor did they bind to the NH-terminal angiotensin I (ANG I) peptide. Little or no binding was detected to angiotensinogens in human, monkey, rat, rabbit, sheep or bovine serum. Mixtures of the clones and analysis of the immune complexes by PAGE indicated that different binding sites on different P-angiotensinogen were detected by some of the monoclonals, while the same or competing sites were recognized by others. No combination of clones tested significantly increased the amount of P-angiotensinogen bound. We interpret these findings to indicate that monoclonal antibodies to 'purified' pig P-angiotensinogen recognize species-specific minor epitope subsets of the protein, but not antigenic determinants common to all.

Biologic effects of transdermal estradiol.

We conducted a dose-response study in 23 postmenopausal women to compare the physiologic effects of transdermal estradiol and oral conjugated equine estrogens. The doses studied were 25, 50, 100, and 200 micrograms of transdermal estradiol per 24 hours, and 0.625 and 1.25 mg of oral conjugated estrogens. Transdermal estradiol increased circulating concentrations of estradiol and estrone. Oral conjugated estrogens also raised the levels of estrogen, particularly estrone. Both preparations lowered gonadotropin levels, decreased the percentages of vaginal parabasal cells, increased the percentage of superficial cells, and lowered urinary calcium excretion. The effects of 0.625 and 1.25 mg of oral estrogens were similar to those of 50 and 100 micrograms of transdermal estradiol per 24 hours, respectively. Oral estrogens significantly increased circulating levels of renin substrate, sex-hormone-binding globulin, thyroxine-binding globulin, and cortisol-binding globulin; transdermal estradiol had no effect. The higher dose of oral estrogens had favorable effects on concentrations of low-density and high-density lipoproteins, but transdermal estradiol did not. Neither preparation affected any of the four clotting factors studied. These data indicate that transdermal estradiol can elicit many of the desirable actions of estrogen while avoiding the pharmacologic effects of oral estrogens on hepatic proteins.

An osmometric method for the bioassay of vasotocin and related peptides in the toad bladder.

This study describes a new method for quantitating the antidiuretic activity of 8-arginine vasotocin (AVT) and related peptides on the isolated toad urinary bladder. The method is based on measuring changes in the osmolality of the surface fluid film of bladders that have been filled with a dilute solution and suspended in humidified air. Eight microliters of Ringer's fluid containing a known concentration of hormone are applied to small paper discs (0.7 cm in diameter), and the discs are then placed with fine forceps onto the outer surface of the bladder to which they adhere. The hormone increases the permeability to water of the epithelium that is underneath the area of the disc, and as water moves from the interior of the bladder along its osmotic gradient to the outer surface of the bladder, the Ringer's fluid in the disc becomes diluted. The magnitude of this dilution is quantitated by removing the discs to a vapor pressure osmometer at timed intervals. A supramaximal dose of AVT reduced disc fluid osmolality by 188 mosmol/kg H2O within 15 min. Similar maximal responses were observed with 8-arginine vasopressin (AVP), oxytocin, and 8-lysine vasopressin, although the potencies of these hormones diminished in the order listed above. AVT was 112-fold more potent than AVP, and AVP, in turn, was 329-fold more potent than 8-lysine vasopressin. The lower limit at which AVT was detected in this assay was 0.25 pg/disc (3 X 10(-11) M). The intra- and interassay variabilities for AVT were 14% and 28% (+/- SD), respectively. This assay is suitable for measuring the biological activity of hormone analogs lacking vasopressor activity, such as desmopressin, which was found to have a hydroosmotic activity of 8.3 +/- 2.4 U/mg. After osmotic stimulation, AVT was detected in toad plasma at a concentration of 1.4 X 10(-10) M. Therefore, this method has the requisite sensitivity for measuring this hormone in biological fluids of amphibia, reptiles, fish, and birds.

Sodium-potassium ATPase in deoxycorticosterone-salt hypertension: opposing effects of sodium load and mineralocorticoids.

Previous studies of the sodium-potassium pump in the deoxycorticosterone (DOC)-salt (DS) model of hypertension yielded contrasting results, some investigators reporting increased and others finding decreased pump activity. To test the possibility that the net pump activity in the DS rats results from separate effects of sodium overload and mineralocorticoid activity, we compared the Na+-K+-ATPase pump in DS rats with that in other experimental models in which these potential determinants do not coincide. Renocortical and myocardial ATPase activities were measured in control rats; adrenalectomized-saline-repleted rats; adrenalectomized aldosterone- or dexamethasone-repleted rats; uninephrectomized, saline-drinking rats; and uninephrectomized, saline-drinking, DOC- and salt-treated rats. DOC- and salt-treated rats had higher (P less than 0.001) blood pressures and lower (P less than 0.05) serum potassium levels than control rats. Renocortical and myocardial ATPase activities were considerably (P less than 0.01) decreased in adrenalectomized, saline-repleted rats, but could be at least partially restituted by either aldosterone or dexamethasone therapy. Uninephrectomized, saline-drinking rats had reduced (P less than 0.01) renocortical and myocardial ATPase activities compared with control rats. In uninephrectomized, saline-drinking rats treated with DOC, renocortical and myocardial ATPase activities were not different from control values. The results of this study suggest that the Na+-K+-ATPase pump in DOC- and salt-treated rats is modulated by the opposing effects of sodium overload-associated suppression and DOC-mediated stimulation.

Heterogeneity of renin substrate released from hepatocytes and in brain extracts.

Renin substrate was characterized in incubation medium of isolated hepatocytes, plasma, and brain extracts of the rat by isoelectric focusing and polyacrylamide gel electrophoresis. The isoelectric focusing (IEF) profile of renin substrate released into incubation medium of rat hepatocytes demonstrated two peaks with isoelectric points (pI) of 4.1 (minor peak) and 4.6 (major peak). Extracts of normal rat brain also showed two forms (pI 4.6 major form, and pI 5.1 minor form). In contrast, normal rat plasma contained a single broad peak of substrate with pI 4.5. On polyacrylamide gel electrophoresis (PAGE), the hepatocytes medium and brain extracts contained forms of substrate with reduced mobility as compared to the plasma form. Intraperitoneal injection of 17 beta estradiol (1 mg) or bilateral nephrectomy significantly elevated renin substrate levels in plasma and increased its release from hepatocytes, however, no change in the IEF or PAGE profiles was evident. There was no remarkable change of substrate concentration in the brain following these treatments. Molecular weights of renin substrate were 60,000-65,000 from all preparations. It remains to be established whether the different forms of renin substrate from hepatocytes represent precursor forms of circulating plasma substrate. The presence of distinct forms of brain renin substrate and the lack of an increase in brain renin substrate following nephrectomy or estrogen treatment suggest local synthesis and support the postulate of an independent renin-angiotensin system in the central nervous system.

Estrogen replacement therapy by transdermal estradiol administration.

To determine whether the nonoral administration of estradiol (E2) might provide physiologic replacement without alteration of hepatic function, 20 postmenopausal women were studied before and after 3 weeks of treatment with either E2-containing transdermal therapeutic systems or placebo. Twenty premenopausal women were also studied. With E2-containing systems, serum E2 and estrone levels were restored to the premenopausal range. Variable responses of the different biochemical and biologic markers of the actions of E2 were observed. The most sensitive marker was vaginal cytology, with the E2 dosage reverting the maturation index to premenopausal values. Hot flashes, measured objectively, were reduced in frequency but not abolished. Serum levels of follicle-stimulating hormone and luteinizing hormone were lowered but remained higher than the premenopausal range. No significant changes were noted in urinary calcium/creatinine and hydroxyproline/creatinine ratios, which were used as markers of bone resorption. With active systems, no significant changes were noted in the concentrations of the hepatic proteins renin substrate and thyroxine-binding globulin or in the binding capacities of cortisol-binding globulin and sex hormone-binding globulin. These results indicate that transdermal E2 administration may be used to provide estrogen replacement while exerting limited effects on hepatic function.

Biological effects of various doses of vaginally administered conjugated equine estrogens in postmenopausal women.

To determine whether vaginal administration of conjugated equine estrogens (VCE) could provide physiological replacement while avoiding effects on hepatic function, as occurs with oral administration, a study was conducted in which 20 postmenopausal women were evaluated before and after the vaginal administration of CE. The dosages studied were 0.3, 0.625, 1.25, and 2.5 mg/day for 4 weeks. Twenty premenopausal women were also studied, and their values were presumed to reflect normal physiological function. The findings in the postmenopausal women were compared with previously reported results obtained in a similar group of subjects given oral CE (OCE). Vaginal cytology returned to premenopausal values with 0.3 mg VCE. This response was similar to that exerted with 1.25 mg OCE. Stepwise increases in circulating estrone and estradiol occurred with increasing dosages. The 2.5-mg dosage of VCE raised estrone levels to values similar to those in premenopausal women in the late follicular phase, and estradiol concentrations were similar to early follicular phase concentrations. Limited or no responses of the systemic markers of estrogen action occurred with all doses of VCE. Small decreases in LH and FSH levels occurred, but no dosage significantly reduced the level of either gonadotropin. Although the urinary calcium to creatinine ratio was significantly reduced by the two largest dosages of VCE, the effect of the 2.5-mg dosage was less than that observed with 0.625 mg OCE, the lowest dosage that protects against osteoporosis. Hepatic protein synthesis was significantly increased only by the higher dosages tested. No dosage had a significant effect on circulating levels of triglycerides or total or fractionated cholesterol levels. These data suggest that the vaginal administration of CE exerts mainly a local effect, with limited or no measurable changes in systemic markers of the action of estrogen.

An increase in high-molecular weight renin substrate associated with estrogenic hypertension.

We have previously reported that estrogens have the potential to induce new forms of renin substrate in addition to elevating the major circulating form of this protein. One of these estrogen-induced forms had a molecular weight in excess of 150,000. In this study we have compared the plasma concentration of the high-molecular-weight renin substrate in normotensive women receiving estrogen therapy and women with estrogenic hypertension. A statistically significant elevation of this protein was associated with estrogenic hypertension and normotensive pregnant women at term. This form of renin substrate differed from the major form with respect to electrophoretic mobility, isoelectric point, and immunologic cross-reactivity. In addition, kinetic analysis indicated that this high-molecular-weight substrate has a significantly higher affinity for the enzyme renin than the major circulating form (Km = 1800 +/- 290 versus 3520 +/- 260 ng angiotensin I equivalents/ml). These results suggest that in addition to renin substrate concentration, substrate composition may play an important role in blood pressure regulation.

PIP: This study investigates whether qualitative rather than quantitative differences in renin substrate were associated with estrogen induction of hypertension by comparing the plasma concentration of high molecular weight renin substrate (HMS) in 18 healthy normotensive, nonpregnant women aged 35-50 taking no medication; 20 normotensive subjects receiving estrogens as oral contraceptives (OCs) or ethinyl estradiol (EE) 50 mcg; and 5 women on OCs or EE 50 mcg who became hypertensive on estrogen therapy. A significant increase in renin substrate was evident in all women with elevated plasma estrogen levels. The difference in total renin substrate levels of normotensive and hypertensive subjects was not statistically significant. HMS differed from the normal molecular weight substrate (NMS) in electrophoretic mobility, isoelectric point, and immunologic cross-reactivity. Kinetic analysis indicated that it also had a significantly higher affinity for the enzyme renin than the major circulating form (Km=1800 +or- 290 versus 3520 +or- 260 ng angiotensin I equivalents/ml). The results suggest that substrate composition may play an important role in blood pressure regulation.

Effect of glutaraldehyde on hydrosmotic response of toad bladder to vasopressin.

The present study investigates the time-, dose-, and temperature-dependence of glutaraldehyde action on the permeability to water of the toad bladder. Bladders preincubated with increasing concentrations of glutaraldehyde become progressively desensitized to the hydrosmotic action of vasopressin (ADH), theophylline, and dibutyryl adenosine 3',5'-cyclic monophosphate (dibutyryl cAMP). The ADH response was reduced by 50% with 0.03% glutaraldehyde applied to the serosal side for 10 min at 4 degrees C. Sixfold higher doses of glutaraldehyde were required with mucosal application. Bladders partially fixed with low-dose glutaraldehyde exhibit a markedly prolonged duration of action of ADH. Bladders fixed with higher doses of glutaraldehyde in the presence of ADH retain a high permeability to water for prolonged periods even in the absence of ADH. This action of glutaraldehyde to stabilize the hormone-induced water channels is also considerably more effective with serosal than with mucosal application. As the rate-limiting permeability barrier for water affected by ADH is known to be located in the apical membrane, these findings suggest that glutaraldehyde exerts its action from an intracellular position. It is postulated that glutaraldehyde stabilizes the ADH-induced channels by cross-linkage of amino groups and other reactive sites at the cytoplasmic surface of the apical membrane and/or by inactivating the intracellular machinery responsible for the dispersal or removal of water channels in the hormone target cell.

Comparison of pharmacodynamic properties of various estrogen formulations.

A group of 23 healthy postmenopausal women received one or more 2-week courses of daily administration of the following estrogen preparations: piperazine estrone sulfate (Ogen), 0.3, 0.625, 1.25, 2.5, and 5.0 mg; micronized estradiol (Estrace), 1, 2, and 10 mg; conjugated estrogens (Premarin), 0.3, 0.625, 1.25, and 2.5 mg; ethinyl estradiol (Estinyl), 10 and 20 micrograms; and diethylstilbestrol, 0.1 and 0.5 mg. Each dosage of each formulation was ingested by three women. In those women who received more than one dosage, each course was separated by a drug-free interval of at least 4 weeks. Pretreatment and posttreatment levels of follicle-stimulating hormone (FSH), luteinizing hormone (LH), corticosteroid-binding globulin-binding capacity, sex hormone-binding globulin-binding capacity, angiotensinogen, estrone, and estradiol were determined. The relative potency of these five estrogen formulations was determined by parallel line analysis for each of these responses, except LH. On a weight basis, piperazine estrone sulfate and micronized estradiol were equipotent for all responses. Conjugated estrogens suppressed FSH in a fashion equipotent to that of the other nonsynthetic estrogens; however, for all three hepatic parameters, the response was exaggerated twofold to threefold. The synthetic estrogens, diethylstilbestrol and ethinyl estradiol, were relatively more potent on a weight basis for every response and produced the most marked response (fourfold to eighteenfold in excess of their FSH suppression) for the hepatic parameters.

Vasopressin resistance induced by low sodium and high mannitol in toad bladder.

The natriferic and hydroosmotic responses to vasopressin (ADH) of the isolated toad bladder were studied as a function of the serosal bath NaCl concentration. Bladders were preincubated in Ringer's solution containing equiosmotic substitutions of mannitol, choline chloride, or choline sulfate for NaCl. Their hydroosmotic and natriferic responses to ADH were measured in NaCl Ringer's solution. Bladders previously bathed on their serosal surface with mannitol had a markedly diminished hydroosmotic response, whereas the natriferic response of these bladders was essentially normal. Similarly, preincubation with either choline chloride or sulfate also inhibited the hydroosmotic response, although the decline was substantially less than that with mannitol. Preincubation with mannitol also inhibited the hydroosmotic response to dibutyryl cAMP. Therefore, the isosmotic substitution of serosal bath NaCl with mannitol decreases primarily the hydroosmotic response to ADH at a post-AMP step. The natriferic effect of the hormone remains essentially intact under these conditions. Different compartments (or cell types) appear to regulate the action of ADH on sodium and on water transport in this tissue because of the selective effects of preincubation with low NaCl Ringer's solution on the hydroosmotic and natriferic responses to ADH.

Multiple forms of human plasma renin substrate.

The objective of this investigation was to determine whether heterogeneity of plasma renin substrate could be observed in states of steroid excess and various forms of hypertensive disease. In states of stimulated renin substrate production by estrogens or glucocorticoids, multiple forms of renin substrate were apparent when stimulation was excessive. Stimulation of substrate production caused by uremia associated with hypertension showed similar results. None, or only trace quantities of the additional forms of renin substrate were evident in subjects with normal or suppressed levels of plasma renin substrate. The additional forms of renin substrate could be distinguished from the normal form on the basis of cross-reactivity with a specific antiserum to the normal form, electrophoretic mobility, and kinetic rate constants. Differences in rate constants of the various forms of plasma renin substrate may account for the altered rate of the renin reaction associated with several states of hypertension. In plasma of patients with renovascular hypertension, significant quantities of a protein which cross-reacted with the antiserum but could not generate angiotensin I were observed.