Defective renal adenylate cyclase response to prostaglandin E2 in spontaneously hypertensive rats

We activated three known components of the adenylate cyclase system in renal membranes from spontaneously hypertensive (SHR) and Wistar-Kyoto (WKY) rats. The basal adenylate cyclase activity and responses to plasma membrane receptor activation by parathyroid hormone, isoproterenol and vasopressin were not different between the two strains. The response to prostaglandin E2 (PGE2), however, was less in the SHR than in the WKY at five, (P less than 0.05), 12 (P less than 0.01) and 16 (P less than 0.01) weeks of age. Activation of either the guanosine-5'-triphosphate (GTP) binding regulatory protein (N) with sodium fluoride (NaF) and guanyl-5'-yl-imidodiphosphate [Gpp(NH)p], or the catalytic unit with manganese chloride (MnCl2) or forskolin were not different between the two groups. When the medullary and cortical plasma membrane adenylate cyclase responses were studied separately, the observed decreased response to PGE2 (of SHR) was found to be entirely in the cortex. Also, the NaF response was reduced in the cortical region of the 12-week-old rats, a finding suggesting a possibility of a post receptor defect. These results show that there is a defective renal adenylate cyclase response specific to prostaglandin E2 in SHR. This defect could be related to the development of hypertension, by changing the natriuretic and/or renal vasodilating effects of these prostaglandins.     

Impaired prostaglandin E(2)/prostaglandin I(2) receptor-G(s) protein interactions in isolated renal resistance arterioles of spontaneously hypertensive rats

The protective effect of vasodilator agents linked to the cAMP pathway is less effective for buffering the vasoconstrictor effect of angiotensin II in young animals with genetic hypertension. To determine the underlying cellular mechanism, experiments were performed on freshly isolated preglomerular resistance arterioles obtained from kidneys of 7-week-old spontaneously hypertensive rats (SHR) and normotensive Wistar-Kyoto rats (WKY). Specific high-affinity saturable binding of (3)H-prostaglandin (PG) E(2) revealed 1 receptor class in renal microvessels; PGE(2) receptor density was similar in SHR and WKY (106 versus 115 fmol/mg; P>0.8), as was receptor affinity (3.6 versus 3.5 nmol/L; P>0.7). Basal cAMP activity was similar in renal arterioles from SHR and WKY. A major finding was that PGE(2), PGI(2), and isoproterenol produced weaker stimulation of cAMP formation in arteriolar cells of SHR (P<0.02). In contrast, GTPgammas and forskolin stimulated cAMP generation to a similar degree in both rat strains, which suggests normal adenylate cyclase activity in hypertension-prone SHR. Immunoblots revealed the presence of 3 classes of G proteins (G(s), G(i), and G(q)) in preglomerular arterioles. The relative amounts of discernible G-protein alpha-subunits in renal resistance vessels did not differ between SHR and WKY. These results extend previous in vivo studies of abnormal renal vascular reactivity in SHR and more directly localize defective coupling of the prostaglandin and beta-adrenergic receptors to a stimulatory G protein and cAMP production in freshly isolated preglomerular arteriolar cells of young SHR. This dysfunction may be due to an abnormal interaction between prostaglandin receptors and G(s) protein that leads to inefficient coupling of initiating steps in the cAMP-protein kinase A cascade during the development of hypertension.     

Reduction in the activity of the stimulatory guanine nucleotide-binding protein in the myocardium of spontaneously hypertensive rats

The beta-adrenergic receptor-adenylate cyclase system of the cardiac membranes in spontaneously hypertensive rats (SHR) 14 weeks old was studied. The maximal activity of the catalytic unit of adenylate cyclase stimulated by purified stimulatory guanine nucleotide-binding protein (Ns) or forskolin was higher in SHR than in control Wistar-Kyoto (WKY) rats. However, adenylate cyclase activity stimulated by isoproterenol and GTP was the same between SHR and WKY rats. Although there was no difference in the amount of Ns which was measured by cholera toxin-catalyzed ADP-ribosylation, the functional activity of Ns in cholate-extracted membranes from SHR was significantly lower than that from WKY rats. There were no strain differences in the number and affinity of beta-adrenergic receptors; the function and amount of the inhibitory guanine nucleotide-binding protein (Ni), and the amount of beta gamma-subunits of Ns and Ni. These findings showed that there is an abnormal signal transduction in this system in SHR due to a reduction in the functional activity of alpha-subunits of Ns.     

Cross-talk between cardiac kappa-opioid and beta-adrenergic receptors in developing hypertensive rats

The kappa-opioid receptor exerts a negative modulatory action on the beta-adrenoceptor and the action is blunted in adult spontaneously hypertensive rats (SHR). In order to determine whether the blunted negative modulation of the beta-adrenoceptor by the kappa-opioid receptor contributes to the development of hypertension, the electrically induced intracellular calcium ([Ca2+]i) transient was measured in single ventricular myocytes of SHR at 4, 6, 8 and 13-week-old and the age-matched Wistar Kyoto (WKY) rats. The electrically induced [Ca2+]i transients were augmented by norepinephrine (NE), a beta-adrenoceptor agonist, over four-fold in WKY rats of all ages studied and in SHR of 4 and 6 weeks of age. The enhancing effect of NE in 8- and 13-week-old SHR was, however, only approximately three-fold, significantly lower than the corresponding values in age-matched WKY rats. Similarly, the electrically induced [Ca2+]i transients were also augmented by forskolin, an activator of adenylate cyclase, by approximately two-fold in WKY rats of all ages and SHR aged 4 and 6 weeks. In SHR aged 8 and 13 weeks, the effect of forskolin was only 1.5-fold, significantly lower than the two-fold increase in the corresponding WKY rats. The enhancing effects of NE and forskolin were attenuated by U50,488H, a selective kappa-opioid agonist, by approximately 50 and 25%, respectively, in both types of rats of all ages studied, with the exception of 13-week-old rats. In rats of this age group, the attenuations by U50,488H on the enhancing effects of NE and forskolin were 17 and 9% in SHR, respectively, significantly less than the corresponding 54 and 29% in WKY. The fact that attenuation of U50,488H on the enhancing effects of NE and forskolin only occurs in 13-week-old SHR when hypertension has been fully developed indicates that the attenuated inhibitory modulation of kappa-opioid receptor stimulation does not contribute to the initiation of hypertension. Interestingly, the enhancing effects of NE and forskolin on the electrically induced [Ca2+]i transient was attenuated in SHR aged from 8 weeks when the blood pressure was rapidly increasing. The different time courses of altered responses to U50,488H, and NE and forskolin suggest that the attenuated negative modulation of kappa-receptor stimulation on the beta-adrenergic receptor is not due to the signal transduction pathway activated by beta-adrenergic stimulation. In 13-week-old SHR with the arterial blood pressure restored to normal by pharmacological manipulations, the blunted responses to NE, U50,488H and forskolin still occurred, indicating that the altered responses to activation of beta-adrenergic and kappa-opioid receptors and adenylate cyclase are not secondary to hypertension.     

Role of altered G-protein expression in the regulation of myocardial adenylate cyclase activity and force of contraction in spontaneous hypertensive cardiomyopathy in rats.

OBJECTIVE: The question of this study was whether alterations in the inhibitory guanine-nucleotide binding protein alpha-subunits (G(i)alpha) contribute to alterations in adenylate cyclase regulation in hypertensive cardiomyopathy of spontaneously hypertensive rats (SHR). DESIGN AND METHODS: G(i)alpha was measured by pertussis toxin-catalysed 32P-adenosine 5'-pyrophosphate (ADP)-ribosylation and radioimmunochemically by competition of rat myocardial membrane extracts to DS 4 antiserum binding to the 125I-radiolabelled C terminus of retinal transducin alpha (125I-KENLKDCGLF). Cardiac beta-adrenoceptors, m-cholinoceptors as well as isoprenaline-, guanine-nucleotide [Gpp(NH)p]- and forskolin-stimulated adenylate cyclase activity and inotropic responses to isoprenaline and carbachol were studied in SHR and age-matched Wistar-Kyoto (WKY, control) rats. RESULTS: In native membranes of SHR there was an increase in pertussis toxin substrates, but a larger increase in the presence of non-ionic detergent Lubrol PX. The radioimmunological quantification of G(i)alpha revealed an increase in membrane extracts of SHR. In addition, myocardial beta-adrenoceptors and myocardial m-cholinoceptors were reduced in SHR compared with in WKY rats. Basal adenylate cyclase, isoprenaline-, Gpp(NH)p- and forskolin-stimulated adenylate cyclase activities were also reduced. However, in the presence of 5 mmol/l MnCl2 no differences in adenylate cyclase activities between SHR and WKY rats were detected under either condition. CONCLUSIONS: The present study shows that the amount of G(i)alpha-proteins and not only pertussis toxin substrates are increased in membranes of hypertrophic hearts from SHR without heart failure. The results obtained with pertussis-labelling depended strongly on the substrate quality of G(i)alpha. Increased G(i)alpha expression and reduced beta-adrenoceptor number might have functional relevance in the regulation of adenylate cyclase activity and force of contraction in SHR. An increase in G(i)alpha expression might play a pathophysiological role, not only in terminal heart failure, but also in hypertrophic cardiomyopathy.     

Myocardial Adrenergic Receptors and Adenylate Cyclase in the Developing Spontaneously Hypertensive Rat

Cardiac membrane preparations from developing spontaneously hypertensive rats (SHR) and Wistar-Kyoto (WKY) rats (0 to 125 days of age) were analyzed for the apparent numbers of alpha-and beta-adrenergic receptors and adenylate cyclase activities in an attempt to correlate biochemical changes with the reported functional changes occurring with the development of hypertension in the SHR. Although the apparent number of alpha- and beta-adrenergic receptors were similar in both strains of rats, isoproterenol-stimulated adenylate cyclase activities were significantly higher (P<0.05) in the prehypertensive SHRs when compared to WKY rats and declined to lower values as hypertension appeared. The percent stimulation produced by isoproterenol remained similar in cardiac membranes from normotensive WKY rats at all ages of development whereas this percent stimulation was 40% higher at birth in the SHRs and declined to approximately one half the original value by 100 days of age (P<0.05). The elevated adenylate cyclase activity observed during the prehypertensive state may contribute to the genesis of hypertension.     

Decreased responsiveness to beta-adrenoceptor agonists in arterial strips from spontaneously hypertensive rats is not associated with alterations in beta-adrenoceptors.

Beta-adrenoceptors in femoral and mesenteric arteries from 13-week-old spontaneously hypertensive rats (SHR) and normotensive Wistar-Kyoto (WKY) rats were studied using radioligand binding assays and were compared with in vitro measurements of beta-adrenoceptor-mediated relaxation. The relaxant responses to noradrenaline via beta-adrenoceptors were significantly decreased in the SHR femoral artery when compared with the WKY femoral artery. However, under the same conditions, arterial relaxant responses to forskolin, an activator of adenylate cyclase, were not significantly different between SHR and WKY rats. Specific binding of 125I-iodocyanopindolol to membranes prepared from femoral arteries of SHR and WKY rats was saturable and of high affinity. Neither the equilibrium dissociation constant of 125I-iodocyanopindolol, nor the maximum number of binding sites were significantly different between SHR and WKY rats. Similar results were obtained in the case of mesenteric arteries from SHR and WKY rats. These results indicate that the decreased responsiveness to beta-adrenoceptor stimulation in SHR arteries is not associated with alterations in beta-adrenoceptors and further support the hypothesis that a reduced function of the stimulatory guanosine triphosphate-binding protein is responsible for the decreased responsiveness to a variety of receptor agonists whose mechanism of action involves adenylate cyclase activation.     

Vascular angiotensin II receptors in SHR

We investigated the density (Bmax) of angiotensin II (ANG II) receptors in the mesenteric vascular bed of spontaneously hypertensive rats (SHR) and age-matched Wistar-Kyoto (WKY) control rats. In 12-week-old SHR, the Bmax and the dissociation constant (Kd) of ANG II binding sites were not different from those of WKY rats in the sodium replete state or after sodium depletion. In prehypertensive (4- and 6-week-old) SHR, the Bmax of the vascular ANG II receptors was significantly higher (p less than 0.05) than in age-matched WKY rats. This result could not be attributed entirely to differences in the circulating renin-angiotensin-aldosterone system in 4-week-old-rats. In 6-week-old WKY rats, the plasma renin activity was significantly higher (p less than 0.05), which may account in part for the higher density of ANG II binding sites in SHR. There was an age-related decrease in the number of ANG II receptors in SHR. The increased density of vascular ANG II receptors in young SHR may play a role in the development of high blood pressure in this model of spontaneous hypertension. The higher number of ANG II binding sites in young SHR is not selective for ANG II receptors, since an increased density of alpha 1-adrenergic receptors was also found in the mesenteric arteries of 4-week-old SHR.     

Enhanced myocardial adenylate cyclase activity in spontaneously hypertensive rats.

This study was designed to clarify the state of beta-adrenergic signal transduction and the disordered level of its transduction in hypertensive hearts, using myocardium from spontaneously hypertensive rats (SHR) as a generic model of essential hypertension. Beta-adrenergic receptor binding sites and dissociation constants in the extracted membranes of adult (70-100 days of age) SHR heart were not significantly different from those of Wistar-Kyoto (WKY) rats, the non-hypertensive control. The adenylate cyclase activities stimulated by isoproterenol with GTP, NaF and forskolin were significantly higher in SHR compared to those in WKY. To determine whether differences in signal transduction are natural or are a result of hypertension, we evaluated chronotropic responses in cultured cells of fetal hearts which had not been exposed to hypertension. Fetal cardiac muscle cells of SHR were more sensitive than WKY to isoproterenol stimulation over a wide concentration range. However, there were no statistically significant differences between these two strains with respect to the density of binding sites. These results suggest that in the transduction of adrenergic signals, alterations distal to the beta-receptors are present in the adult hearts of hypertensive rats, and, that the adrenergic signal transduction is already exaggerated in the pre-hypertensive fetal stage.     

Effects of chronic ethanol ingestion on the vasoactive intestinal peptide receptor-effector system from rat seminal vesicle membranes

We studied the modifications of the vasoactive intestinal peptide (VIP) receptor/effector system from the rat seminal vesicle after chronic ethanol ingestion. Ethanol treatment resulted in a decreased height of the secretory epithelium of seminal vesicle as well as in a weight loss of this gland. These morphological changes were accompanied by an increase of immunoreactive vasoactive intestinal peptide (VIP) levels and a decrease of the stimulatory effect of VIP adenylate cyclase activity in the seminal vesicle. The loss of sensitivity of the enzyme to VIP was conceivably related to a decrease in the affinity of VIP receptors rather than to a decrease in their number. The changes in the affinity of the VIP receptors were accompanied with a lower sensitivity of VIP binding to GTP, which suggest an uncoupling between the receptor and the transductor molecules. However, chronic exposure to ethanol did not modify either the levels of G-protein subunits (alpha(s) and alpha(i1/2)) or the GTPase activity from seminal vesicle membranes. Moreover, ethanol feeding did not affect adenylate cyclase activity stimulated by forskolin or by Gpp(NH)p. Thus, ethanol-induced changes in the sensitivity of adenylate cyclase to VIP appear to be attributed to an alteration in the VIP-receptor/G-protein interphase rather than in the G-protein/adenylate cyclase connection.     

Stimulation by forskolin of the thyroid adenylate cyclase, cyclic AMP accumulation and iodine metabolism

Forskolin, a diterpene hypotensive drug, activates adenylate cyclase in brain and in some other tissues (Seamon et al., 1981). Forskolin activated adenylate cyclase in particulate preparations and enhanced cyclic AMP accumulation in slices of dog thyroid. These effects were maximal within minutes and remained constant afterwards. The action of forskolin on intact cells disappeared rapidly after washing. It reproduced two known cyclic AMP-mediated TSH effects: the activation of secretion and of protein iodination. Forskolin thus provides a very convenient tool for the study of the action of defined elevations of cyclic AMP level in thyroid cells. The activation by forskolin of adenylate cyclase was not reduced by Mn2+ which uncouples TSH and PGE1 action. This suggests that in the thyroid also, forskolin acts beyond the receptor level. The effect of forskolin on cyclic AMP accumulation was inhibited by the known negative regulators of this system in the thyroid, acetylcholine, iodide, norepinephrine, PGF1 alpha and adenosine. On the other hand, forskolin potentiated the effects of TSH, PGE1 and cholera toxin. These data show that, though it does not require the receptors for its action, forskolin does not uncouple them from the catalytic unit of adenylate cyclase.     

Renal dopamine receptor signaling mechanisms in spontaneously hypertensive and Fischer 344 old rats

Dopamine plays an important role in the regulation of renal sodium excretion. The activation of D1-like receptors located on the proximal tubules causes inhibition of tubular sodium reabsorption by inhibiting Na,H-exchanger and Na,K-ATPase activity. The D1-like receptors are linked via G proteins to the multiple cellular signaling systems namely adenylyl cyclase and phospholipase C (PLC). A defective renal dopamine receptor function exists in spontaneously hypertensive rats (SHR). In the proximal tubules of SHR, the stimulation of adenylyl cyclase and PLC caused by dopamine was significantly reduced in comparison with Wistar-Kyoto (WKY) rats. Also unlike the effects seen in WKY, D1-like receptor activation did not inhibit Na,K-ATPase and Na,H-exchanger activities in SHR. In addition, reduced quantity of Gq/11alpha proteins was detected in the basolateral membranes of SHR compared to WKY rats. Studies revealed that there may be a primary defect in D1-like receptors leading to an altered signaling system in the proximal tubules and reduced dopamine-mediated effect on renal sodium excretion in SHR. Recently, it has been shown that the disruption of D1A receptors at the gene level causes hypertension in mice. Similar to SHR, dopamine and D1-like receptor agonist failed to inhibit Na,K-ATPase activity in the proximal tubules of old Fischer 344 rats. Unlike the observations in SHR where D1-like receptors were equal to WKY rats, there is a 50% decrease in D1-like receptor number in basolateral membranes of the old rats compared to the adult rats. Dopamine was unable to stimulate G proteins in the basolateral membranes of old rats compared to the adult rats. It is suggested that a defective dopamine receptors/signaling system may contribute to the development and maintenance of hypertension. Also, the inability of dopamine to inhibit Na,K-ATPase may lead to a reduced renal sodium excretion in response to dopamine in old rats.     

Vascular atrial natriuretic factor receptors in spontaneously hypertensive rats.

OBJECTIVE: The aim was to investigate vascular receptors for atrial natriuretic factor (ANF) in spontaneously hypertensive (SHR), Wistar-Kyoto (WKY), and Wistar rats (WR) at different ages. METHODS: Relaxation and guanylate cyclase responses of blood vessels to atrial natriuretic factor were investigated, as was the binding of 125I-ANF to vascular membranes and ANF receptor subtypes, using sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) in reducing conditions, after solubilisation and irreversible binding of 125I-ANF. RESULTS: Vascular relaxation responses of aorta showed an increased sensitivity to ANF in four week old SHR [pD2 = 8.9 (SEM 0.1) v 8.5(0.1) in WKY rats, p < 0.05] while sensitivity was similar for the three strains at older ages. Production of cyclic GMP in mesenteric arteries in response to 100 nmol.litre-1 ANF was greater (p < 0.05) in SHR than in WKY rats at four weeks of age, but was similar in older rats. The density of binding sites for ANF in mesenteric arteries, however, was lower in SHR at four weeks (p < 0.01), and increased in older rats, becoming similar to that of normotensive rats at 12 weeks of age. Affinity of ANF sites was similar in all strains. The proportion of high and low molecular weight ANF binding peptides in solubilised blood vessel membranes on SDS-PAGE was similar in all strains except in four week old SHR, in which binding to the high molecular weight band (presumably the guanylate cyclase containing receptor) was increased relative to the low molecular weight band (non-cyclase-coupled receptor) in comparison to other strains and ages. CONCLUSIONS: Activity of guanylate cyclase in response to occupancy of ANF receptors may be increased in young SHR. Normal relaxation of blood vessels in response to ANF in older SHR could result in failure to counteract the increased vasoconstrictor activity present in these rats, which could play a role in the increase in blood pressure.     

Autonomic receptor interactions in isolated cardiac myocytes from hypertensive rats

Isolated ventricular myocytes from adult (16 to 20 weeks) spontaneously hypertensive (SHR) and normotensive (WKY) rats were utilized to examine adrenergic and cholinergic receptor expression and interaction. Binding assays were performed using quinuclidinyl benzilate (QNB) and iodocyanopindolol (ICYP) for cholinergic and beta-adrenergic receptors, respectively. In addition, cAMP was measured as an index of adrenergic-cholinergic control of adenylate cyclase. Data from radioligand binding experiments indicated that muscarinic cholinergic receptors were depressed (22%) in SHR myocytes, while beta-adrenergic receptor density was comparable to that of WKY myocytes. Heterologous receptor modulation in isolated myocytes as assessed by displacement analysis with and without guanosine 5'-triphosphate (GTP), showed that carbachol displacement of QNB was shifted five fold to the right in the presence of GTP and that the beta-adrenergic agonist isoproterenol did not prevent the GTP-mediated binding alteration. In contrast, carbachol modulated the GTP-shift of ICYP displacement by isoproterenol and these effects were comparable in both WKY and SHR myocytes. Furthermore, the ability of carbachol to blunt the stimulation of adenylate cyclase by isoproterenol was also comparable in myocytes isolated from adult SHR and control animals. Thus, the observed decrement in muscarinic cholinergic receptor expression did not alter adrenergic-cholinergic interactions as assessed by displacement assays using guanine nucleotides, or the control of cAMP levels. In addition, isolated myocytes provide a useful system for analyzing receptor expression and regulation and how these parameters may be altered in the hypertensive heart.     

Sodium responsiveness of central alpha 2-adrenergic receptors in spontaneously hypertensive rats

The responsiveness of central nervous system alpha 2-adrenergic receptors in the neural control of renal function was compared in conscious spontaneously hypertensive rats (SHR) and normotensive Wistar-Kyoto rats (WKY) on normal or high sodium intake (3-4 weeks of 1% NaCl for drinking). The responsiveness of central alpha 2-adrenergic receptors was determined by comparing among groups the dose-response curves for the effects of cumulative intracerebroventricular injections of guanabenz (5, 25, and 125 micrograms) on changes in mean arterial pressure, renal sympathetic nerve activity, and urinary sodium excretion. Guanabenz altered mean arterial pressure similarly in SHR on normal or high sodium intake and in WKY on normal or high sodium intake. High sodium intake shifted the guanabenz-renal sympathetic nerve activity and guanabenz-urinary sodium excretion dose-response curves to the left in SHR and to the right in WKY. The dose-response curves between SHR and WKY on normal sodium intake were similar. Surgical renal denervation or pretreatment with an alpha 2-adrenergic receptor antagonist (rauwolscine, 30 micrograms i.c.v.) attenuated the ability of guanabenz to inhibit renal sympathetic nerve activity or increase urinary sodium excretion in SHR and WKY on either normal or high sodium intake. We conclude that the responsiveness of central nervous system alpha 2-adrenergic receptors regarding the neural control of renal function is increased by high sodium intake in conscious SHR, but not in conscious normotensive WKY.     

Parathyroid hormone desensitization in renal membranes of vitamin D-deficient rats is associated with a postreceptor defect

We examined the characteristics of PTH resistance in vitamin D-deficient rats employing renal membranes in vitro. Homologous desensitization was characterized by diminished PTH-stimulated adenylate cyclase activity and was associated with a reduction in PTH-binding capacity, but not affinity. Heterologous desensitization was also seen, as manifested by decreased calcitonin (CT)-stimulated adenylate cyclase activity with normal CT receptor binding. The reduced capacity of the nonhormonal effectors NaF and guanylylimidodiphosphate to stimulate adenylate cyclase indicated a postreceptor defect at the level of the guanyl nucleotide-binding protein (G protein), whereas a normal forskolin response was consistent with a fully functional catalytic component. The G protein deficiency was confirmed by demonstrating that the addition of extracts of vitamin D-sufficient membranes to preparations of vitamin D-deficient membranes restored the normal responses to NaF and guanylylimidodiphosphate. In addition, cholera toxin- and pertussis toxin-catalyzed labeling of vitamin D-deficient renal membranes with [32P]NAD revealed a decrease in both the stimulatory and inhibitory binding proteins. Experiments with testicular membranes in vitro indicated that the adenylate cyclase abnormality was absent in tissue lacking PTH receptors. The results suggest that a major contribution to PTH resistance in vitamin D-deficient animals is a postreceptor defect at the level of the G proteins and that this defect is manifest only in tissue expressing the PTH receptor.     

Differential roles of high and low affinity guanosine 5'-triphosphate binding sites in the regulation of follicle-stimulating hormone binding to receptor and signal transduction in bovine calf testis membranes

We have previously shown that FSH receptors are physically and functionally associated with a guanine nucleotide regulatory protein (Gs) in membranes of calf testis. Using N-ethylmaleimide (NEM), forskolin, and cholera toxin as probes, we have investigated the role of low and high affinity GTP-binding sites of stimulatory guanine nucleotide-binding protein of adenylate cyclase (Gs) in the activation of adenylate cyclase. When calf testis membranes were exposed to NEM (1 mM), FSH binding to receptors was slightly (30%) decreased, but the receptors showed continued sensitivity to GTP, resulting in a further decrease in [125I]human FSH binding to receptors. Pretreatment of membranes with NEM (up to 20 microM) produced no effect on GTP-binding. A dose-dependent decrease in high affinity GTP-binding sites, however, was observed at higher (greater than 50 microM) NEM. Adenylate cyclase activity was reduced in response to GTP gamma S or NaF concomitant to a decrease in high affinity GTP-binding sites in membranes treated with 50-100 microM NEM, or completely abolished in membranes exposed to 300 microM NEM. Stimulation by forskolin indicated that the significant inhibition of adenylate cyclase activity occurring in membranes exposed to low NEM (50-100 microM) was not due to inactivation of catalytic unit of adenylate cyclase by NEM. Pretreatment of membranes with 100 micrograms/ml cholera toxin and NAD slightly (18%) reduced specific FSH binding but did not affect Gpp(NH)p-binding. However, adenylate cyclase stimulation by GTP plus FSH in these membranes was significantly enhanced. When membranes were treated with higher concentration of cholera toxin (250 micrograms/ml), the adenylate cyclase stimulation by GTP plus FSH was abolished due to uncoupling of FSH receptors from Gs and a significant decrease in high affinity GTP-binding sites. Our results suggest that high affinity GTP-binding sites of Gs coupled to FSH receptors are essential for FSH and guanine nucleotide activation of adenylate cyclase. The low affinity binding sites bind GTP and thereby regulate FSH binding but are not involved in the activation of adenylate cyclase.     

Effects of dietary sodium on brain angiotensin II receptors in spontaneously hypertensive rats

The effects of dietary sodium on the characteristics of angiotensin II (A II) receptor sites in the hypothalamus-thalamus-septum-midbrain (HTSM) region were examined in spontaneously hypertensive rats (SHR) and Wistar-Kyoto rats (WKY). Twenty-four SHR and 24 WKY were divided into two groups respectively, which were maintained on high sodium diets or low sodium diets for 4 weeks, respectively. The binding capacity and affinity of the A II receptors were measured by radioreceptor assay. In WKY, the binding capacity of the A II receptors in the high sodium group was significantly lower than that in the low sodium group. On the other hand, the binding capacity of A II receptors in the high sodium group was significantly lower than that in the low sodium group. On the other hand, the binding capacity of A II receptors was not significantly different between high and low sodium groups in SHR. The secretion of arginine vasopressin (AVP) increased significantly in SHR with high sodium intake. The present results suggest that in WKY the decrease of the binding capacity of the A II receptors in the HTSM region in response to a high sodium intake serves to attenuate an osmotical stimulus to AVP secretion. However, in SHR such a regulatory mechanism as adjusting the binding capacity of the A II receptors is lacking, and this seems to be responsible, at least in part, for the enhanced secretion of AVP on the sodium loading.     

Forskolin effects on the cAMP system and steroidogenesis in the immature rat ovary

The diterpene forskolin was found to activate the adenylate cyclase system in intact tissue and membrane preparations of the immature rat ovary. The cyclic AMP (cAMP) response reached a maximal level after 5 min and no decline was observed even after 4 h of incubation. Forskolin stimulated production of both progesterone and testosterone in a pattern similar to that produced by luteinizing hormone (LH) or dibutyryl-cAMP (dbcAMP). In combination with LH, follicle-stimulating hormone (FSH) or prostaglandin E2 (PGE2), forskolin potentiated the hormone effects on adenylate cyclase activity in membrane preparations. Pretreatment with LH or PGE2 desensitized the cells to further hormone stimulation, while the forskolin response was unaffected. Pre-exposure to forskolin did not desensitize the cells to a subsequent stimulation by LH or PGE2. The presence of 8-bromo-cAMP (brcAMP) in the preincubation medium reduced the subsequent hormone response. These results demonstrate a rapid and sustained activation of the adenylate cyclase system by forskolin in the rat ovary. The steroidogenic response was similar to that of known stimulators of ovarian cells (LH, dbcAMP). The inability of forskolin to induce desensitization of the adenylate cyclase system demonstrates, however, important differences between hormone and non-hormone activation. Consequently, forskolin can be a useful tool for investigation of the mechanisms involved in the desensitization process.     

Adipocyte responsiveness to norepinephrine in spontaneously hypertensive rats

The present study describes the effect of norepinephrine on lipolysis and adenylate cyclase activity in adipocytes from Wistar-Kyoto (WKY) and spontaneously hypertensive (SH) rats. The adipocytes were incubated in the presence of norepinephrine (10(-7) to 10(-4) mol/L) and the lipolytic activity was measured according to the accumulation of glycerol after one hour incubation. The results showed that norepinephrine induced a lower lipolytic activity in adipocytes from SH rats. cAMP-phosphodiesterase activities in adipocyte homogenates from WKY and SH rats were the same for both preparations. The effect of norepinephrine (10(-7) to 10(-4) mol/L) on adenylate cyclase activity in fat cell membranes from SH rats was decreased compared with WKY fat cell membranes. Adenylate cyclase activities in the presence of 10 mmol/L NaF were the same in both preparations. beta-receptor characteristics were examined, and the data demonstrate a statistically significant decrease in beta-receptor density in fat cell membranes from SH rats. The dissociation constants (Kd) were the same for WKY and SH preparations. This article suggests that adipocyte responsiveness to norepinephrine is decreased in SH rats. The decreased response to norepinephrine may be explained by a lower beta-receptor density in fat cell membranes from SH rats.