Compartmentation of cyclic adenosine 3',5'-monophosphate signaling in caveolae.

The cAMP-signaling pathway is composed of multiple components ranging from receptors, G proteins, and adenylyl cyclase to protein kinase A. A common view of the molecular interaction between them is that these molecules are disseminated on the plasma lipid membrane and random collide with each other to transmit signals. A limitation to this idea, however, is that a signaling cascade involving multiple components may not occur rapidly. Caveolae and their principal component, caveolin, have been implicated in transmembrane signaling, particularly in G protein-coupled signaling. We examined whether caveolin interacts with adenylyl cyclase, the membrane-bound enzyme that catalyzes the conversion of ATP to cAMP. When overexpressed in insect cells, types III, IV, and V adenylyl cyclase were localized in caveolin-enriched membrane fractions. Caveolin was coimmunoprecipitated with adenylyl cyclase in tissue homogenates and copurified with a polyhistidine-tagged form of adenylyl cyclase by Ninitrilotriacetic acid resin chromatography in insect cells, suggesting the colocalization of adenylyl cyclase and caveolin in the same microdomain. Further, the regulatory subunit of protein kinase A (RIIalpha, but not RIalpha) was also enriched in the same fraction as caveolin. Gsalpha was found in both caveolin-enriched and non-caveolin-enriched membrane fractions. Our data suggest that the cAMP-signaling cascade occurs within a restricted microdomain of the plasma membrane in a highly organized manner.

Accumulation of molecules involved in alpha1-adrenergic signal within caveolae: caveolin expression and the development of cardiac hypertrophy.

OBJECTIVE: Caveolin, a major protein component of caveolae, is now considered to be an inhibitor of cellular growth and proliferation. In this study, we examined the localization of the molecules involved in alpha1-adrenergic receptor signal relative to that of caveolin in the heart and the changes in caveolin expression during the development of hypertrophy in SHR. METHODS: We purified the caveolar protein fractions from rat cardiac tissues, H9C2 cells, and rat vascular smooth muscle cells. Using radioligand receptor binding assay and immunoblot analysis, we examined the distribution and the amount of alpha1-AR and caveolin. RESULTS: Caveolin-3, the alpha1-adrenergic receptor, Gq and PLC-beta subtypes (PLC-beta1, -beta3) were found exclusively in the caveolar fraction in the above tissues. Caveolin-3 were co-immunoprecipitated with alpha1-adrenergic receptor and Gq from the cardiac tissues. The amount of caveolin subtypes expression (caveolin-1 and -3) and the amount of the alpha1-adrenergic receptor were examined in the hearts of SHR and age-matched WKY (4- and 24-weeks-old). The amount of caveolin-3 expression was significantly smaller in SHR at 24-weeks-old than that in SHR at 4-weeks-old and that in WKY at 24-weeks-old. CONCLUSIONS: The molecules involved in alpha1-adrenergic signaling are confined to the same microdomain as caveolin. A decrease in caveolin-3 expression may play a role in the development of cardiac hypertrophy in SHR, presumably through de-regulating the inhibition of growth signal in the hearts of SHR in the hypertrophic stage.

Inhibition of adenylyl cyclase by caveolin peptides.

Caveolae and their principal component caveolin have been implicated in playing a major role in G protein-mediated transmembrane signaling. We examined whether caveolin interacts with adenylyl cyclase, an effector of G protein signaling, using a 20-mer peptide derived from the N-terminus scaffolding domain of caveolin-1. When tissue adenylyl cyclases were examined, cardiac adenylyl cyclase was inhibited more potently than other tissue adenylyl cyclases. The caveolin-1 peptide inhibited type V, as well as type III adenylyl cyclase, overexpressed in insect cells, whereas the same peptide had no effect on type II. The caveolin-3 scaffolding domain peptide similarly inhibited type V adenylyl cyclase. In contrast, peptides derived from the caveolin-2 scaffolding domain and a caveolin-1 nonscaffolding domain had no effect. Kinetic studies showed that the caveolin-1 peptide decreased the maximal rate (Vmax) value of type V without changing the Michaelis constant (Km) value for the substrate ATP. Studies with various truncations and point mutations of this peptide revealed that a minimum of 16 amino acid residues and intact aromatic residues are important for the inhibitory effect. The potency of inhibition was greater when adenylyl cyclase was in stimulated condition vs. basal condition. Thus, caveolin may be another cellular component that regulates adenylyl cyclase catalytic activity. Our results also suggest that the caveolin peptide may be used as an isoform-selective inhibitor of adenylyl cyclase.

Association analysis of restriction fragment length polymorphism for alpha 2-adrenergic receptor genes in essential hypertension in Japan.

Recently, restriction fragment length polymorphism (RFLP) of alpha 2-adrenergic receptor gene (alpha 2-C10) digested with Bsu36I restriction enzyme has been reported in US populations. Therefore, we examined the association of this RFLP with essential hypertension by comparing the frequency of specific alleles for this gene in Japanese populations. The distribution of this RFLP was compared with that in US populations. Subjects were hypertensive patients with a family history of essential hypertension (n = 56) and normotensive subjects whose parents had no history of essential hypertension (n = 46). DNA was prepared from leukocytes. RFLP was determined by use of Southern blot analysis with an alpha 2-C10 probe and Bsu36I. The frequencies of the major (12-kb) and minor (5.8-kb) alleles were 0.30 and 0.70 in hypertensive patients and 0.38 and 0.62 in normotensive subjects, respectively. The difference between observed alleles in all subjects in each group was not significant (chi 2 = 1.33, P > .1). The difference between the overall allelic frequency in Japan and that reported in US populations was significant. This study found no evidence for an association between alpha 2-adrenergic receptor gene/Bsu36I RFLP and essential hypertension in Japan. However, the findings showed that the allele frequency in Japan differed from that reported in US populations.

Angiotensinogen gene polymorphism near transcription start site and blood pressure: role of a T-to-C transition at intron I.

Molecular variants of the angiotensinogen gene, a key component of the renin-angiotensin system, are considered genetic risk factors for primary hypertension. A relation between the angiotensinogen gene locus and hypertension has been found in whites, Japanese, and African Caribbeans but not in Chinese. The lack of a consistent association between M235T polymorphism at exon 2 and hypertension has suggested that another site in linkage disequilibrium with M235T is the causal mutation. We studied the relations among plasma angiotensinogen concentrations, blood pressure, related clinical variables, and mutations of the 5' upstream core promoter region of the human angiotensinogen gene in 274 subjects recruited from our outpatient clinic. We confirmed that plasma angiotensinogen concentration was significantly correlated with A-20C mutation and percent body fat and found that systolic and diastolic blood pressures were significantly correlated with G-6A and T+68C mutations. These results suggest that mutations near the transcription start site may be associated with increased blood pressure.

Regulation of type V adenylyl cyclase by PMA-sensitive and -insensitive protein kinase C isoenzymes in intact cells.

Abstract Type V adenylyl cyclase (AC) was stably over-expressed in HEK293 cells (293AC-V). Forskolin-stimulated cAMP accumulation in 293AC-V was 5 times as great as that in control cells. PMA, a protein kinase C (PKC) activator, enhanced cAMP accumulation in 293AC-V cells dose-and time-dependently and this enhancement was abolished by staurosporine. Insulin also enhanced cAMP accumulation in 293AC-V cells. Co-transfection of PKC-zeta, but not PKC-alpha, potentiated the effects of insulin. These data suggest that type V AC activity is regulated in cells by PKC isoenzymes through different extracellular stimuli.

Isoform-dependent activation of adenylyl cyclase by proteolysis.

Recent findings have suggested that the cellular proteolytic system plays a major role in the regulation of various intra- and extra-cellular signaling. It was previously shown that proteolytic treatment of adenylyl cyclase leads to the activation of this enzyme. We demonstrate that this activation occurs in an adenylyl cyclase isoform-dependent manner. The type II isoform was strongly activated (approximately 500%), the type III isoform was modestly activated (approximately 30%),and the type V isoform was inhibited by trypsin. Activation of type II adenylyl cyclase occurred in trypsin dose- and time-dependent manners and was blocked by a trypsin inhibitor in a dose-dependent manner. Other proteases, such as thrombin and plasminogen, similarly activated the type II isoform, but not the others. Our data suggest that proteolytic activation is an isoform- and thus cell type-dependent mechanism of altering adenylyl cyclase catalytic activity.

Effect of dietary sodium on platelet alpha 2-adrenoceptors in young normotensive men with or without a family history of hypertension.

OBJECTIVE: To study the effects of genetics on the response of platelet alpha 2-adrenoceptors to a change in salt intake. METHODS: Biochemical measurements and radioligand binding assays in platelets were performed in 11 normotensive male university students with a family history of essential hypertension (FH+) and in 17 students without a family history of hypertension (FH-). The 28 students were fed a high-sodium diet for 7 days and a low-sodium diet for 7 days. RESULTS: In FH+ subjects the number of alpha 2-adrenergic receptors on platelet membrane fractions increased significantly from the high-sodium diet to the low-sodium diet, even though plasma noradrenaline concentrations tended to increase with the low-sodium diet. There was no change in the number of alpha 2-adrenoceptors in the FH--group. In both groups the radioligand binding affinity was decreased during a low-sodium period compared with in a high-sodium period. CONCLUSION: In the FH+ subjects the change in platelet alpha 2-adrenoceptors associated with altered sodium status was similar to that seen in patients with salt-sensitive hypertension, suggesting that there is a genetic susceptibility to sodium.

Participation of the sympathetic nervous system in hypertension in rats with subtotal renal ablation.

To clarify the role of the sympathetic nervous system in the development of hypertension in chronic renal failure, plasma levels and urinary excretions of catecholamines were evaluated in male Sprague-Dawley rats. The renal mass of the rats was reduced by removing one kidney and two-thirds of the contralateral kidney (5/6 nephrectomy). Five-sixths nephrectomy was followed by significant increases in serum creatinine (to 0.55 +/- 0.03 mg/dl) and urea nitrogen (to 42.9 +/- 3.8 mg/dl). There was a concomitant increase in mean blood pressure, measured directly by an implanted aortic catheter, in comparison with control rats (155.3 +/- 8.3 versus 123.6 +/- 3.3 mmHg, P less than 0.01). Both plasma levels and urinary excretion of norepinephrine and epinephrine were elevated in the 5/6-nephrectomized rats compared with controls. Mean blood pressure correlated negatively with 24-h creatinine clearance (r = -0.66, P less than 0.05), and positively with plasma norepinephrine (r = 0.83, P less than 0.01) and urinary excretion of norepinephrine (r = 0.63, P less than 0.05). These results suggest that not only the decrease in renal function, but also hyperactivity of the sympathetic nervous system, may be involved in the pathogenesis of hypertension in rats with subtotal renal ablation.

Antihypertensive effects of 2-octynyladenosine (YT-146), a selective adenosine A2 receptor agonist, in Dahl salt-sensitive rats.

The antihypertensive effects of a novel adenosine A2 receptor agonist, 2-octynyl adenosine (YT-146), were evaluated in Dahl salt-sensitive rats. After rats were fed a high-salt (8% NaCl) diet for 2 or 3 weeks, they received oral YT-146 (0.1 or 1.0 mg/kg) or vehicle as a single dose (acute study) or once daily for 10 days (chronic study). In the acute study, tail-cuff blood pressure (BP) and pulse rate (PR) were measured before and 3, 6, and 24 h after administration, and blood samples were collected 3 h after administration. In the chronic study, BP and PR were measured 3 and 24 h after administration and urine was collected for 24 h on day 9. Blood samples were also collected 3 h after administration on day 10. BP was significantly lowered by 1.0 mg/kg of YT-146 in either the acute study (from 184 +/- 3 to 152 +/- 5 mm Hg, P < .01) or the chronic study (from 226 +/- 4 to 201 +/- 2 mm Hg, P < .01), while an increase in PR was not observed (acute study: from 382 +/- 8 to 366 +/- 3 beats/min; chronic study: from 420 +/- 8 to 411 +/- 8 beats/min). YT-146 had no effect on plasma renin activity (PRA), plasma aldosterone, vasopressin (ADH), and atrial natriuretic peptide (ANP) in the acute study.(ABSTRACT TRUNCATED AT 250 WORDS)

Alpha 1-adrenergic receptors in cardiac ventricles of Dahl rats.

This study was designed to examine the effects of sex, age, and a high-salt diet on cardiac alpha 1-adrenoceptors in an animal model of genetic hypertension, the Dahl salt-sensitive rat. Ventricular alpha 1-adrenoceptors were measured by radioligand binding with [3H]prazosin in membrane fractions in Dahl S and R rats of 7, 12, and 15 weeks of age. In both S and R rats, the maximal binding (Bmax) of alpha 1-adrenoceptor binding was greater in male than in female rats. The Bmax decreased with age in both the S and R strains; at 12 weeks of age, Bmax was approximately one-half of that observed at 7 weeks of age in both S and R strains. In the rats fed a high-salt diet, the Bmax tended to be greater in S rats than in R rats at 12 weeks of age and this difference became significant at 15 weeks of age. A significant positive correlation was found between the Bmax and the heart-to-body weight ratio in the Dahl S and R rats. The dissociation constant (Kd) was not different between male S and R rats at each age. These results suggest that the ventricular alpha 1-adrenoceptor may be involved in cardiac hypertrophy in Dahl rats.

Enhanced inhibitory effect of alpha 2-adrenoceptor stimulation on the formation of cAMP in glomeruli of spontaneously hypertensive rats.

Renal alpha 2-adrenoceptors are known to be increased in spontaneously hypertensive rats (SHR) compared with Wistar-Kyoto rats (WKY). To investigate whether this difference affects the second messenger system, we examined the effect of alpha 2-adrenoceptor stimulation on the formation of cAMP in microdissected glomeruli and proximal convoluted tubules obtained from the kidneys of SHR and WKY. The formation of glomerular cAMP, which was stimulated by parathyroid hormone (PTH), was inhibited by alpha 2-adrenoceptor stimulation. In contrast, the inhibitory effect of alpha 2-adrenoceptor stimulation on PTH-induced cAMP formation in proximal convoluted tubules was not significantly different between SHR and WKY. These results confirm the inhibitory action of alpha 2-adrenoceptors on the formation of cAMP in glomeruli and proximal tubules and suggest that the greater inhibitory effect on glomerular cAMP formation in SHR may reflect an increase in alpha 2-adrenoceptor density in SHR kidneys.

[Fundamental and clinical studies of flomoxef in the pediatric field].

Fundamental and clinical studies of flomoxef (FMOX, 6315-S) were conducted and the obtained results are summarized as follows. For the pharmacokinetic investigation, FMOX at 10 or 20 mg/kg was administered by intravenous drip infusion over 30 minutes. In the 10 mg/kg group, the maximum blood concentration was reached just after completion of the drip infusion with the mean concentration of 30.3 +/- 4.5 micrograms/ml, and the mean half-life was 0.734 +/- 0.196 hour. The 6-hour urinary excretion rate was 72.3%. In the 20 mg/kg group, which also showed the peak concentration immediately after completion of the drip infusion, the mean peak blood concentration was 54.3 +/- 9.7 micrograms/ml and the mean half-life was 0.628 +/- 0.185 hour. The 6-hour urinary excretion rate was 69.3%. The urinary recovery tended to be lower in children than in adults. Between 10 mg/kg and 20 mg/kg, a definite correlation was observed between dose levels and blood concentrations. In the clinical investigation conducted with a total of 30 patients (22 with respiratory tract infection, 3 with lymphadenitis, 2 each with urinary tract infection and cellulitis, and 1 with acute osteomyelitis), FMOX was found to be excellent in 14 cases, good in 9, fair in 1, poor in 1, and not evaluable in 5. The efficacy rate was, therefore, 92.0%. In the bacteriological evaluation, 8 out of 12 clinically isolated strains were eradicated, 2 unchanged and 2 unknown. The elimination rate was 80.0%. Regarding side effects, no abnormal clinical symptoms were observed. As abnormal laboratory values, eosinophilia, prolongation of APTT, increased platelet count, and a slight elevation of GOT were observed.

[Catecholamine and dopamine].

Almost all the genes of the enzymes which synthesize and metabolize the catecholamines (dopamine, norepinephrine, epinephrine) have been cloned and the gene targeting technology have been applied to introduce the gene knockout mouse such as thyrosine hydroxylase and dopamine beta hydroxylase. At least nine adrenergic receptors and five dopamine receptors have been cloned, which include alpha 1A-, alpha 1 B-, alpha 1 D-, alpha 2 A-, alpha 2B-, alpha 2C-, beta 1-, beta 2-, beta 3-adrenergic receptors and D1-, D2-, D3-, D4-, D5-dopamine receptors. Transgenic mouse as well as gene knockout mouse of these genes have been also produced. Furthermore, intracellular signal transduction systems of the catecholamines have been clarified using molecular techniques, including nine subtypes of adenylyl cyclase. Using these cloned genes and transgenic and gene knockout mouse, more detailed features of the catecholamine systems and those receptors and intracellular signal transduction systems will be clarified in near future.

[Adenosine and adenosine receptors in the kidney].

In the kidney, adenosine plays important regulatory roles, including renal blood flow, glomerular filtration rate, renin secretion, tubuloglomerular feedback, tubular reabsorption of sodium and water, sympathetic neurotransmitter release, and erythropoietin secretion. These functions are mediated through adenosine 1 (A1)-receptors and adenosine 2 (A2)-receptors. These receptors couple to the inhibition and stimulation of adenylate cyclase, through Gi and Gs proteins, respectively. A variety of other effecter systems have been reported to be coupled to A1 receptors, including phospholipase C, phospholipase A2 and potassium, as well as Ca++ channels. Recently, A1 receptors, A2 receptors and novel A2 receptor have been cloned, sequenced and expressed. In association with the development of selective adenosine analogues, we are now ready to take up problems at the biochemical and molecular biological levels.