Charles O. Watlington

β-Adrenergic stimulation of frog skin mucous glands: Non-specific inhibition by adrenergic blocking agents

Abstract 1. 1. In frogs s.c. norepinephrine (NE) produced an increase in volume of skin secretions (primarily mucous gland in origin) and increases in pH, [Na+], [Cl−], and [HCO3−]. The volume response was linear to the lof of NE dose in the range of 50–400 μg. per frog. 2. 2. Aminophylline mimicked the effect of NE. 3. 3. An α- and a β-adrenergic blocking agent both inhibited the effects of norepinephrine and aminophylline, indicating non-specific blockade. 4. 4. Isoproterenol produced a greater volume of skin secretions than the weaker β-adrenergic agent phenylephrine. 5. 5. The results suggest that the catecholamine increase in mucous secretion is β-adrenergic and may be mediated by increased tissue levels of cyclic 3′,5′-AMP.

Renal corticosterone 6β-hydroxylase in the spontaneously hypertensive rat

Abstract Excess 6β-OH-corticosterone production by family 3A cytochromes P-450 may play a role in genesis of hypertension in the spontaneously hypertensive rat (SHR), by producing a renal defect in Na + excretion. Renal cytochromes P-450 may be a causal factor in this genetic model. Since family 3A P-450 is present in rat kidney (collecting duct), the renal family 3A catalytic (6β-OHase) and immunoreactive activities were compared in SHR and normotensive control (Wistar-Kyoto; WKY) rats. Corticosterone 6β-hydroxulation is markedly higher in SHR than in WKY renal microsomal preparations. Western blot analysis with antibodies to rat and rabbit liver family 3A isoforms demonstrated related proteins. Densitometry revealed greater relative intensity of staining in SHR compared to WKY with both antibodies. Both antibodies inhibited corticosterone 6β-hydroxylation by SHR renal microsomes. Increased renal 6β-OH-corticosterone production by increased renal family 3A cytochromes P-450 may play a role in the blood pressure elevation in SHR.

α-Adrenergic inhibition of Na+ transport: The interaction of vasopressin and 3′,5′-AMP

Abstract It has been proposed that α-adrenergic inhibition of Na+ transport across isolated frog skin is mediated by a decrease in adenyl cyclase activity and a resultant decrease in 3′,5′-AMP synthesis. To test this hypothesis the interaction of phenylephrine, predominantly an α-adrenergic substance, with vasopressin and 3′,5′-AMP was studied in the presence of propranalol, a β-blocker. The short-circuit current (Is) technique was used. Phenoxybenzamine, an α-adrenergic blocking agent, prevented the phenylephrine-induced decrease in Is. Dose-response studies indicated the minimum and maximum effective concentrations to be approx. 1 and 100 μM, respectively. A submaximal concentration of phenylephrine (2.5 μM) decreased Is in the presence of a low vasopressin concentration (1 munit/ml), but the effect was prevented by a higher vasopressin concentration (100 munits/ml). The higher vasopressin concentration did not prevent an Is decrease by the maximal effective phenylephrine concentration (0.1 mM). These findings suggest that vasopressin and α-adrenergic stimulation affect the same Na+ transport regulatory system. In the presence of 0.1 mM phenylephrine, vasopressin did not alter Is. However, a concentration of 3′,5′-AMP which was equipotent or less increased Is in the presence of the same phenylephrine concentration. The findings are compatible with the hypothesis that α-adrenergic stimulation decreases adenyl cyclase activity and 3′,5′-AMP synthesis in the frog-skin epidermis which in turn mediates a decrease in Na+ permeability and net Na+ transport.

Augmented arterial pressure responses to cyclosporine in spontaneously hypertensive rats. Role of cytochrome P-450 3A.

Evidence to support a hypertensinogenic role of family 3A cytochrome P-450 (CYP3A) activity is that troleandomycin, a selective inhibitor of CYP3A, decreases both blood pressure and in vivo corticosterone 6 beta-hydroxylation in spontaneously hypertensive rats (SHR). Renal CYP3A activity is markedly increased in SHR compared with Wistar-Kyoto (WKY) rats. Cyclosporine acutely increases both systolic blood pressure and renal total cytochrome P-450 in SHR. We tested the hypothesis that the augmentation of blood pressure by cyclosporine is mediated by a further increase in renal CYP3A activity. Accordingly, we assessed the effect of troleandomycin administration on cyclosporine-induced systolic blood pressure increase and renal and hepatic microsomal CYP3A activity in SHR. Cyclosporine (5 mg/kg SC) given daily in 11-week-old SHR resulted in substantial augmentation of blood pressure after 6 days. This blood pressure increase was attenuated by troleandomycin (40 mg/kg) given either during or after development of hypertension. Cyclosporine increased renal (60%) but decreased hepatic (25%) microsomal CYP3A activity in SHR. In contrast, cyclosporine failed to produce any detectable increase in either blood pressure or renal CYP3A activity in WKY rats. Troleandomycin completely inhibited renal CYP3A activity measured after cyclosporine treatment of SHR, which correlated with its attenuation of the cyclosporine-induced blood pressure increase. These findings suggest that renal CYP3A could play an important role in acute cyclosporine-induced hypertension.

Chloride flux across frog skins of low potential difference

Abstract In a series of unselected isolated skins of Rana pipiens in summer, with a mean potential difference (PD) of 13 mV (5–30 mV), the short-circuit current equivalent was significantly less than net Na+ flux. No discrepancy was found in absence of chloride (skins bathed in sulfate Ringers). Net Cl− influx was demonstrated in Cl−-containing Ringers and was of appropriate magnitude to explain the discrepancy. The active transport of Na was quantitatively similar to values seen in the skins of higher PD. Comparison of Cl− flux in this study with mean Cl− flux and PD values from other series suggests an inverse seasonal variation of Cl−. flux and PD.

Calcium flux in isolated frog skin; the effect of parathyroid substances.

Summary Net inward calcium flux occurs across isolated short-circuited frog skin in the absence of a calcium concentration gradient. Calcium influx and calcium outflux are inversely related to the intermittent open-circuit skin PD but are not a function of short-circuit current. The changes in calcium flux in the hour after PTE administration suggest that PTE increases influx and decreases outflux. PTE and PTH do not alter short-circuit current or skin PD.

Efect of adrenergic stimilation on ion transport across skin of living frogs

Abstract 1. 1. The influence of the intravenous administration of catecholamines and a β adrenergic blocking agent on short-circuit current (Is) skin resitance and sodium flux was studied with living anesthetized frog. 2. 2. Epinephrine (α plus β adrenergic stimulation) adn isoproterenol (β stimulation) produced an increase of Is, which was not explained by sodium transport changes, and mucous release. 3. 3. In contrast to epinephrine, isoproterenol decreased skin resistance and increased sodium outflux. 4. 4. Pronethalol (a β adrenergic blocking agent) decreased I3, net sodium flux, and increased skin resistance. 5. 5. The results indicate that ion transport across the skin, in vivo, is influenced by α and β adrenergic stimulation. They support the concept that the two stimuli produce opposing effects on sodium transport, i.e. α stimulation decreases and β stimulation increases sodium permeability of the skin.

Maternal environment defines blood pressure and its response to troleandomycin in spontaneously hypertensive rats

Relationship between family-3A cytochrome P-450-dependent (troleandomycin inhibitable) and maternal environmental-dependent systolic blood pressure (SBP) was investigated in spontaneously hypertensive rats (SHR). Adult SHR nursed by foster or natural SHR mothers had indistinguishable SBP. Troleandomycin reduced 50% of Wistar-Kyoto (WKY)-SHR strain difference in SBP. SHR having WKY foster mothers had SBP similar to troleandomycin-reduced SHR levels, which was unaffected by troleandomycin. The two components of SBP elevation appear identical. Because observations of others demonstrated that WKY fostered to SHR show no SBP increase, the maternally dependent/troleandomycin-sensitive component of SBP elevation may reflect epistatic interaction between genes determining maternal differences and offspring sensitivity, respectively.

Synthesis of 6β-hydroxyaldosterone by A6 (toad kidney) cells in culture

Abstract Incubation of aldoslerone with confluent layers of A6 (toad kidney) cells leads to its hydroxylation at the 6β-position. 6β-Hydroxyaldosterone is the major metabolite when the incubation is carried out at pH 6.8, whereas the product comprises dβ-hydroxy-17-isoaldosterone accompanied by some 6β-hydroxyapoaldosterone at pH 7.4. All products were identified by high-field 1 H nuclear magnetic resonance spectroscopy. Control experiments indicated that the side-chain isomerization to form the 17-iso and apo derivatives occurs after the cytochrome P 450-dependent synthesis of 6β-hydroxyaldosterone. (Steroids 55 :482–487, 1990)

Regulation of sodium transport by alteration of chloride conductance

Abstract A previously undescribed in vivo Na+ transport regulatory mechanism is demonstrated in skin of frogs. This adaptive response, produced by preconditioning in high salt environment, is mediated by decrease in passive Cl− permeability or conductance. It is independent of aldosterone suppression and of the presence of Na+ in the conditioning medium.