Atrial natriuretic factor: radioimmunoassay and effects on adrenal and pituitary glands

A simple and sensitive radioimmunoassay was developed for measurement of immunoreactive atrial natriuretic factor (IR-ANF) in rat and human plasma and in rat atria. The two atria contain about 20 micrograms ANF per rat. The right atrium contained 2.5 times more ANF than did the left. Ether anesthesia and morphine markedly increased IR-ANF in rat plasma. The concentration of IR-ANF in plasma of clinically normal human subjects was 65.3 +/- 2.5 pg/ml. Paroxysmal tachycardia and rapid atrial pacing significantly increased IR-ANF in human plasma. Two- to seven-fold higher concentrations were found in coronary sinus blood than in the peripheral circulation. In the plasma of rats and humans, circulating ANF is probably a small-molecular-weight peptide. ANF acts on the adrenal and the pituitary. ANF inhibits aldosterone secretion from rat zona glomerulosa and steroid secretion by bovine adrenal zona glomerulosa and fasciculata. ANF stimulated the basal secretion of arginine vasopressin (AVP) in vitro and inhibited KCl-stimulated release of AVP.     

Adenosine regulates the release of adrenocorticotropic hormone (ACTH) from cultured anterior pituitary cells

We have recently shown the presence of adenosine receptors coupled to adenylate cyclase in anterior pituitary and in the present studies we have investigated the effects of adenosine on ACTH release. The R-site specific analogs of adenosine such as N-Ethylcarboxamide adenosine (NECA), L-N⁶-phenylisopropyl adenosine (PIA), 2-chloro-adenosine (2-Cl-Ado) all stimulated ACTH release in a dose-dependent manner. NECA was the most potent analog and stimulated ACTH release by about 170% with an apparent Ka of 0.1 µM, whereas PIA and 2-Cl-Ado were less potent and stimulated the release by about 110% and 125% with an apparent Ka of 0.2 and 0.4 µ-M respectively. The stimulation of ACTH release by NECA was inhibited by 3-isobutyl-1-methylxanthine (IBMX). On the other hand, adenosine deaminase (ADA) treatment of the cells also stimulated ACTH release as well as adenylate cyclase activity by about 2-fold, suggesting that endogenous adenosine plays an inhibitory role in the release of ACTH. Other agents, such as corticotropin-releasing factor (CRF), vasoactive intestinal peptide (VIP) and forskolin (FSK) also stimulated ACTH release from these cells. In addition, the stimulation by an optimal concentration of NECA was almost additive with maximal stimulation caused by VIP and FSK. These data suggest that adenosine modulates ACTH release from anterior pituitary through its interaction with adenosine receptors coupled to adenylate cyclase.Abbreviations NECA N-Ethylcarboxamideadenosine - PIA L-N⁶-Phenylisopropyladenosine - 2-Cl-Ado 2-chloroadenosine - FSK Forskolin - VIP Vasoactive Intestinal Peptide - CRF Corticotropin Releasing Factor - ADA Adenosine Deaminase - IBMX 3-Isobutyl-1-methylxanthine     

Modulation of adenylate cyclase activity by Ca2+, phospholipid-dependent protein kinase in rat brain striatum

Summary The effects of purified Ca²⁺, phospholipid-dependent protein kinase (C-kinase) were studied on adenylate cyclase activity from rat brain striatum. C-kinase treatment of the membranes stimulated adenylate cyclase activity, the maximal stimulation between 50–80% was observed at 3.5 U/ml, whereas the catalytic subunit of cAMP dependent protein kinase did not show any effect on enzyme activity. The inclusion of Ca²⁺ and phosphatidyl serine did not augment the percent stimulation of adenylate cyclase by C-kinase, however EGTA inhibited the stimulatory effect of C-kinase on enzyme activity. Furthermore, the known inhibitors of C-kinase such as polymyxin-B and 1-(5-Isoquinoline sulfonyl)-2-methylpiperazine dihydrochloride (H-7) also inhibited the stimulatory effect of C-kinase on adenylate cyclase activity. In addition, in the presence of GTP the stimulatory effects of C-kinase on basal and N-Ethylcarboxamide adenosine- (NECA-), dopamine-(DA) and forskolin- (FSK) sensitive adenylate cyclase activities were augmented. On the other hand, the inhibitory effect of high concentrations of GTP on enzyme activity was attenuated by C-kinase treatment. In addition, oxotremorine inhibited adenylate cyclase activity in a concentration dependent manner, with an apparent Ki of about 10 µM and C-kinase treatment almost completely abolished this inhibition. These data suggest that C-kinase may play an important role in the regulation of adenylate cyclase activity possibly by interacting with a guanine nucleotide regulatory protein.Abbreviations C-kinase Ca^2– phospholipid-dependent protein kinase - NECA N-Ethylcarboxamide adenosine - DA Dopamine - FSK Forskolin - PMA Phorbol 12-(-Myristate), 13-Acetate, H-7, 1-(5-isoquinoline sulfonyl)-2-methylpiperazine dihydrochloride Presented in part at the VIth International Conference on Cyclic nucleotides, calcium and protein phosphorylation signal transduction in biological systems. September 2-6, 1986, Bethesda, MD (USA).M.B.A.-S. was Canadian Heart Foundation Scholar during the course of these studies.     

Presence of “Ra” and “P”-site receptors for adenosine coupled to adenylate cyclase in cultured vascular smooth muscle cells

The existence of adenosine receptors coupled to adenylate cyclase in cultured vascular smooth muscle cells from rat aorta is demonstrated in these studies. Adenosine, N⁶-phenylisopropyladenosine, adenosine N′-oxide and 2-chloroadenosine stimulated adenylate cyclase in a concentration dependent manner. The stimulation was dependent on the presence of guanine nucleotides and was blocked by 3-isobutyl-1-methylxanthine. In contrast, 2′ deoxyadenosine inhibited adenylate cyclase activity. Adenosine and 2-chloroadenosine showed a biphasic effect on adenylate cyclase, stimulation occurred at low concentrations. The activation of adenylate cyclase by N⁶-phenylisopropyladenosine was also dependent on the Mg²⁺ concentration. The data suggest that vascular smooth muscle cells have both “Ra” and “P” receptors for adenosine, and it can be postulated that the relaxant effect of adenosine on vascular smooth muscle may be mediated by its interaction with “Ra” receptors associated with adenylate cyclase.     

Role of oxidative stress in angiotensin II-induced enhanced expression of Gi(alpha) proteins and adenylyl cyclase signaling in A10 vascular smooth muscle cells

We have previously reported that angiotensin II (ANG II) treatment of A10 vascular smooth muscle cells (VSMCs) increased inhibitory G proteins (G(i) protein) expression and associated adenylyl cyclase signaling which was attributed to the enhanced MAP kinase activity. Since ANG II has been shown to increase oxidative stress, we investigated the role of oxidative stress in ANG II-induced enhanced expression of G(i)alpha proteins and examined the effects of antioxidants on ANG II-induced enhanced expression of G(i)alpha proteins and associated adenylyl cyclase signaling in A10 VSMCs. ANG II treatment of A10 VSMCs enhanced the production of O(2)(-) and the expression of Nox4 and P47(phox), different subunits of NADPH oxidase, which were attenuated toward control levels by diphenyleneiodonium (DPI). In addition, ANG II augmented the expression of G(i)alpha-2 and G(i)alpha-3 proteins in a concentration- and time-dependent manner; the maximal increase in the expression of G(i)alpha was observed at 1 to 2 h and at 0.1-1.0 microM. The enhanced expression of G(i)alpha-2 and G(i)alpha-3 proteins was restored to control levels by antioxidants such as N-acetyl-L-cysteine, alpha-tocopherol, DPI, and apocynin. In addition, ANG II also enhanced the ERK1/2 phosphorylation that was restored to control levels by DPI. Furthermore, the inhibition of forskolin-stimulated adenylyl cyclase activity by low concentrations of 5'-O-(3-triotriphosphate) (receptor-independent G(i) functions) and ANG II-, des(Glu(18),Ser(19),Glu(20),Leu(21),Gly(22))atrial natriuretic peptide(4-23)-NH(2) (natriuretic peptide receptor-C agonist), and oxotremorine-mediated inhibitions of adenylyl cyclase (receptor-dependent functions) that were augmented in ANG II-treated VSMCs was also restored to control levels by antioxidant treatments. In addition, G(s)alpha-mediated diminished stimulation of adenylyl cyclase by stimulatory hormones in ANG II-treated cells was also restored to control levels by DPI. These results suggest that ANG II-induced enhanced levels of G(i)alpha proteins and associated functions in VSMCs may be attributed to the ANG II-induced enhanced oxidative stress, which exerts its effects through mitogen-activated protein kinase signaling pathway.     

Solvent-induced free energy landscape and solute-solvent dynamic coupling in a multielement solute

Molecular dynamics simulations using a simple multielement model solute with internal degrees of freedom and accounting for solvent-induced interactions to all orders in explicit water are reported. The potential energy landscape of the solute is flat in vacuo. However, the sole untruncated solvent-induced interactions between apolar (hydrophobic) and charged elements generate a rich landscape of potential of mean force exhibiting typical features of protein landscapes. Despite the simplicity of our solute, the depth of minima in this landscape is not far in size from free energies that stabilize protein conformations. Dynamical coupling between configurational switching of the system and hydration reconfiguration is also elicited. Switching is seen to occur on a time scale two orders of magnitude longer than that of the reconfiguration time of the solute taken alone, or that of the unperturbed solvent. Qualitatively, these results are unaffected by a different choice of the water-water interaction potential. They show that already at an elementary level, solvent-induced interactions alone, when fully accounted for, can be responsible for configurational and dynamical features essential to protein folding and function.     

Effects of β-adrenoceptor blocker pindolol, calcium antagonist verapamil and their combination on retention in step-down- and shuttle-box-trained rats and on brain biogenic monoamines

The effects of the β-adrenoceptor blocker pindolol and the calcium antagonist verapamil administered alone or in combination on retention in step-down- and shuttle-box-trained rats and on the biogenic monoamine levels in the frontal cortex and hippocampus were examined. The chronic oral treatment with pindolol impaired retention in step-down- and shuttle-box-trained rats, decreasing the dopamine (DA) and noradrenaline (NA) levels and increasing the serotonin (5-HT) levels in the cortex and hippocampus. Verapamil did not influence retention in step-down- and shuttle-box avoidance situation and the biogenic monoamine levels in the frontal cortex and hippocampus. It should, however, be noted that the chronic oral treatment with verapamil completely abolished the retention-impairing effect of pindolol, restoring to normal DA, NA and 5-HT levels. These findings might be of interest to clinical practice and suggest the necessity for using a combination of β-blockers with Ca²⁺ antagonists in case of prolonged treatment of cardiovascular diseases.     

Pregnancy success of lactating Holstein cows after a single administration of a sustained-release formulation of recombinant bovine somatotropin

Background

Results regarding the use of bovine somatotropin for enhancing fertility in dairy cattle are variable. Here, the hypothesis was tested that a single injection of a sustained-release preparation of bovine somatotropin (bST) during the preovulatory period would improve pregnancy success of lactating dairy cows at first service.

Results

The first experiment was conducted in a temperate region of Mexico. Cows inseminated following natural estrus or timed artificial insemination were given a single injection of bST or a placebo injection at insemination (n = 100 cows per group). There was no significant difference between bST and control groups in the proportion of inseminated cows diagnosed pregnant (29 vs 31% pregnant). The second experiment was performed during heat stress in Florida. Cows were subjected to an ovulation synchronization regimen for first insemination. Cows treated with bST received a single injection at 3 days before insemination. Controls received no additional treatment. As expected, bST did not increase vaginal temperature. Treatment with bST did not significantly increase the proportion of inseminated cows diagnosed pregnant although it was numerically greater for the bST group (24.2% vs 17.8%, 124–132 cows per group). There was a tendency (p = 0.10) for a smaller percent of control cows to have high plasma progesterone concentrations (≥ 1 ng/ml) at Day 7 after insemination than for bST-treated cows (72.6 vs 81.1%). When only cows that were successfully synchronized were considered, the magnitude of the absolute difference in the percentage of inseminated cows that were diagnosed pregnant between bST and control cows was reduced (24.8 vs 22.4% pregnant for bST and control).

Conclusion

Results failed to indicate a beneficial effect of bST treatment on fertility of lactating dairy cows.