Boye L. Jensen

Chloride Regulates Afferent Arteriolar Contraction in Response to Depolarization

-Renal vascular reactivity is influenced by the level of dietary salt intake. Recent in vitro data suggest that afferent arteriolar contractility is modulated by extracellular chloride. In the present study, we assessed the influence of chloride on K+-induced contraction in isolated perfused rabbit afferent arterioles. In 70% of vessels examined, K+-induced contraction was abolished by acute substitution of bath chloride. Consecutive addition of Cl- (30, 60, 80, 100, 110, and 117 mmol/L) restored the sensitivity to K+, and half-maximal response was observed at 82 mmol/L chloride. The calcium channel antagonist diltiazem (10(-6) mol/L) abolished K+-induced contractions. Bicarbonate did not modify the sensitivity to chloride. Norepinephrine (10(-6) mol/L) induced full contraction in depolarized vessels even in the absence of chloride. Iodide and nitrate were substituted for chloride with no inhibitory effect on K+-induced contraction. Approximately 30% of the vessels constricted in response to K+ in the absence of chloride. This response was reversibly blocked by the alpha1-blocker phentolamine (PA) (10(-5) mol/L) and, with PA present, the dependence on chloride was similar to the above series. The results show that K+-induced contraction of smooth muscle cells in the afferent arteriole is highly sensitive to chloride, whereas neurotransmitter release and ensuing contraction is not dependent on chloride. Thus, there are different activation pathways for depolarizing vasoconstrictors and for the sympathetic nervous system in renal afferent arterioles. This could be of physiological relevance for the resetting of afferent arteriolar sensitivity during changes in salt intake.

Direct Demonstration of Exocytosis and Endocytosis in Single Mouse Juxtaglomerular Cells

The rate of renin secretion from renal juxtaglomerular (JG) cells is the major determinant of the activity of the renin-angiotensin system. However, the mechanisms involved in the excretion and turnover of secretory granules in the JG cells remain obscure. Therefore, in the present study, the whole-cell patch-clamp technique was applied to single JG cells from the mouse kidney to measure changes in cell membrane capacitance (Cm) as an index of secretory activity. Resting JG cell Cm was stable, on average 3. 13+/-0.13 pF (SEM, n=106). In isotonic solutions, Cm was unaffected by [Cl-]i. Cm was consistently increased (7.0+/-1.3% and 7.2+/-3.1%) by intracellular cAMP (1 to 10 micromol/L). This effect was mimicked by extracellular application of the beta-agonist isoproterenol to the JG cells (9.4+/-3.1%). At 100 micromol/L, cAMP induced a paradoxical decrease in Cm of </=20%, which was mimicked by forskolin. Cell swelling induced by a 7% reduction in osmolality increased Cm with no significant additional effects to [Cl-]i and cAMP. cAMP increased whole-cell outward current 2- to 4-fold in all groups, but no correlation between changes in whole-cell currents and Cm existed. We conclude that the whole-cell patch-clamp method allows the study of exocytosis and endocytosis in JG cells. Renin release induced by the cAMP pathway and by cell swelling is exocytotic, and high-intracellular cAMP levels activate membrane retrieval mechanisms.

Depolarization-induced calcium influx in rat mesenteric small arterioles is mediated exclusively via mibefradil-sensitive calcium channels

In this study, intracellular Ca2+ was measured as the Fura‐2 ratio (R) of fluorescence excited at 340 and 380 nm (F340/F380) in nonpressurized rat mesenteric small arterioles (Ø (lumen diameter) 10–25 μm). The response to depolarization using 75 mM KCl was an increase in R from a baseline of 0.96±0.01 ([Ca2+]i ∼74 nM) to 1.04±0.01 (∼128 nM) (n=80). The response to 75 mM K+ was reversibly abolished in Ca2+‐free physiological saline solution, whereas phentolamine (10 μM) or tetrodotoxin (1 μM) had no effects. LaCl3 (200 μM) inhibited 61±9% of the response. A [K+]‐response curve indicated that the Ca2+ response was activated between 15 and 25 mM K+. The data suggest that the Ca2+ response was caused by the activation of voltage‐dependent Ca2+ channels. Mibefradil use dependently inhibited the Ca2+ response to 75 mM K+ by 29±2% (100 nM), 73±7% (1 μM) or 89±7% (10 μM). Pimozide (500 nM) use dependently inhibited the Ca2+ response by 85±1%. Nifedipine (1 μM) inhibited the Ca2+ response to 75 mM K+ by 41±12%. The response was not inhibited by calciseptine (500 nM), ω‐agatoxin IVA (100 nM), ω‐conotoxin MVIIA (500 nM), or SNX‐482 (100 nM). Using reverse transcriptase–polymerase chain reaction, it was shown that neither CaV2.1a (P‐type) nor CaV2.1b (Q‐type) voltage‐dependent Ca2+ channels were expressed in mesenteric arterioles, whereas the CaV3.1 (T‐type) channel was expressed. Furthermore, no amplification products were detected when using specific primers for the β1b, β2, or β3 auxiliary subunits of high‐voltage‐activated Ca2+ channels. The results suggest that the voltage‐dependent Ca2+ channel activated by sustained depolarization in mesenteric arterioles does not classify as any of the high‐voltage‐activated channels (L‐, P/Q‐, N‐, or R‐type), but is likely to be a T‐type channel. The possibility that the sustained Ca2+ influx observed was the result of a T‐type window current is discussed.

Renin secretion from permeabilized juxtaglomerular cells requires a permeant cation.

Abstract The cytosolic concentration of chloride correlates directly with renin secretion from renal juxtaglomerular granular (JG) cells. In the present study, the mechanism by which chloride stimulates renin release was investigated in a preparation of permeabilized rat glomeruli with attached JG cells. An isosmotic increase in the concentration of chloride by 129 mM stimulated renin release 16- to 20-fold. Substitution of K+ by the impermeant cation N-methyl-d-glucamine (NMDG) abolished this response, while substitution with Na+ caused marginal inhibition. Substitution with Cs+ had no effect. Addition of sucrose, which permeates the secretory granules poorly, also abolished the stimulation of renin secretion by KCl. The response to KCl was not affected by K+-channel antagonists or by agonists of K+ channels. Chloride channel blockers were also without effect on the secretory response to KCl. When the ATP concentration was lowered from 1 to 0.1 mM renin release was stimulated, while an increase in the ATP concentration from 1 to 5 mM had no effect. Blockers of ATP-sensitive (KATP) channels did not modify the response to chloride. The present data suggest that chloride stimulates renin release after entry of KCl into the renin secretory granules which results in swelling and release of renin.