Max Salomonsson

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.

Role of vascular potassium channels in the regulation of renal hemodynamics

K(+) conductance is a major determinant of membrane potential (V(m)) in vascular smooth muscle (VSMC) and endothelial cells (EC). The vascular tone is controlled by V(m) through the action of voltage-operated Ca(2+) channels (VOCC) in VSMC. Increased K(+) conductance leads to hyperpolarization and vasodilation, while inactivation of K(+) channels causes depolarization and vasoconstriction. K(+) channels in EC indirectly participate in the control of vascular tone by several mechanisms, e.g., release of nitric oxide and endothelium-derived hyperpolarizing factor. In the kidney, a change in the activity of one or more classes of K(+) channels will lead to a change in hemodynamic resistance and therefore of renal blood flow and glomerular filtration pressure. Through these effects, the activity of renal vascular K(+) channels influences renal salt and water excretion, fluid homeostasis, and ultimately blood pressure. Four main classes of K(+) channels [calcium activated (K(Ca)), inward rectifier (K(ir)), voltage activated (K(V)), and ATP sensitive (K(ATP))] are found in the renal vasculature. Several in vitro experiments have suggested a role for individual classes of K(+) channels in the regulation of renal vascular function. Results from in vivo experiments are sparse. We discuss the role of the different classes of renal vascular K(+) channels and their possible role in the integrated function of the renal microvasculature. Since several pathological conditions, among them hypertension, are associated with alterations in K(+) channel function, the role of renal vascular K(+) channels in the control of salt and water excretion deserves attention.

Closure of multiple types of K+ channels is necessary to induce changes in renal vascular resistance in vivo in rats

Inhibition of K+ channels might mediate renal vasoconstriction. As inhibition of a single type of K+ channel caused minor or no renal vasoconstriction in vivo in rats, we hypothesized that several classes of K+ channels must be blocked to elicit renal vasoconstriction. We measured renal blood flow (RBF) in vivo in anesthetized Sprague–Dawley rats. Test agents were infused directly into the renal artery to avoid systemic effects. Inhibition of BKCa and Kir channels (with TEA and Ba2+, respectively) caused small and transient reductions in RBF (to 93 ± 2% and 95 ± 1% of baseline, respectively). KATP, SKCa or Kv channel blockade (with glibenclamide, apamin and 4-aminopyridine, respectively) was without effect. However, a cocktail of all blockers caused a massive reduction of RBF (to 15 ± 10% of baseline). Nifedipine and mibefradil abolished and reduced, respectively, this RBF reduction. The effect of the cocktail of K+ channel blockers was confirmed in mice using the isolated blood-perfused juxtamedullary nephron preparation. A cocktail of K+ channel openers (K+, NS309, NS1619 and pinacidil) had only a minor effect on baseline RBF in vivo in rats, but reduced the vasoconstriction induced by bolus injections of norepinephrine or angiotensin II (by 33 ± 5% and 60 ± 5%, respectively). Our results indicate that closure of numerous types of K+ channels could participate in the mediation of agonist-induced renal vasoconstriction. Our results also suggest that renal vasoconstriction elicited by K+ channel blockade is mediated by nifedipine-sensitive Ca2+ channels and partly by mibefradil-sensitive Ca2+ channels.

Connexin mimetic peptides fail to inhibit vascular conducted calcium responses in renal arterioles

Vascular conducted responses are believed to play a central role in controlling the microcirculatory blood flow. The responses most likely spread through gap junctions in the vascular wall. At present, four different connexins (Cx) have been detected in the renal vasculature, but their role in transmission of conducted vasoconstrictor signals in the preglomerular arterioles is unknown. Connexin mimetic peptides were previously reported to target and inhibit specific connexins. We, therefore, investigated whether conducted vasoconstriction in isolated renal arterioles could be blocked by the use of mimetic peptides directed against one or more connexins. Preglomerular resistance vessels were microdissected from kidneys of Sprague-Dawley rats and loaded with fura 2. The vessels were stimulated locally by applying electrical current through a micropipette, and the conducted calcium response was measured 500 mum from the site of stimulation. Application of connexin mimetic peptides directed against Cx40, 37/43, 45, or a cocktail with equimolar amounts of each, did not inhibit the propagated response, whereas the nonselective gap junction uncoupler carbenoxolone completely abolished the propagated response. However, the connexin mimetic peptides were able to reduce dye coupling between rat aorta endothelial cells shown to express primarily Cx40. In conclusion, we did not observe any attenuating effects on conducted calcium responses in isolated rat interlobular arteries when exposed to connexin mimetic peptides directed against Cx40, 37/43, or 45. Further studies are needed to determine whether conducted vasoconstriction is mediated via previously undescribed pathways.

Na+-independent, nifedipine-resistant rat afferent arteriolar Ca2+ responses to noradrenaline: possible role of TRPC channels.

Aim:  In rat afferent arterioles we investigated the role of Na+ entry in noradrenaline (NA)‐induced depolarization and voltage‐dependent Ca2+ entry together with the importance of the transient receptor potential channel (TRPC) subfamily for non‐voltage‐dependent Ca2+ entry.

Role of connexin40 in the autoregulatory response of the afferent arteriole

Connexins in renal arterioles affect autoregulation of arteriolar tonus and renal blood flow and are believed to be involved in the transmission of the tubuloglomerular feedback (TGF) response across the cells of the juxtaglomerular apparatus. Connexin40 (Cx40) also plays a significant role in the regulation of renin secretion. We investigated the effect of deleting the Cx40 gene on autoregulation of afferent arteriolar diameter in response to acute changes in renal perfusion pressure. The experiments were performed using the isolated blood perfused juxtamedullary nephron preparation in kidneys obtained from wild-type or Cx40 knockout mice. Renal perfusion pressure was increased in steps from 75 to 155 mmHg, and the response in afferent arteriolar diameter was measured. Hereafter, a papillectomy was performed to inhibit TGF, and the pressure steps were repeated. Conduction of intercellular Ca(2+) changes in response to local electrical stimulation was examined in isolated interlobular arteries and afferent arterioles from wild-type or Cx40 knockout mice. Cx40 knockout mice had an impaired autoregulatory response to acute changes in renal perfusion pressure compared with wild-type mice. Inhibition of TGF by papillectomy significantly reduced autoregulation of afferent arteriolar diameter in wild-type mice. In Cx40 knockout mice, papillectomy did not affect the autoregulatory response, indicating that these mice have no functional TGF. Also, Cx40 knockout mice showed no conduction of intercellular Ca(2+) changes in response to local electrical stimulation of interlobular arteries, whereas the Ca(2+) response to norepinephrine was unaffected. These results suggest that Cx40 plays a significant role in the renal autoregulatory response of preglomerular resistance vessels.

Role of renal vascular potassium channels in physiology and pathophysiology

The control of renal vascular tone is important for the regulation of salt and water balance, blood pressure and the protection against damaging elevated glomerular pressure. The K+ conductance is a major factor in the regulation of the membrane potential (Vm) in vascular smooth muscle (VSMC) and endothelial cells (EC). The vascular tone is controlled by Vm via its effect on the opening probability of voltage‐operated Ca2+ channels (VOCC) in VSMC. When K+ conductance increases Vm becomes more negative and vasodilation follows, while deactivation of K+ channels leads to depolarization and vasoconstriction. K+ channels in EC indirectly participate in the control of vascular tone by endothelium‐derived vasodilation. Therefore, by regulating the tone of renal resistance vessels, K+ channels have a potential role in the control of fluid homoeostasis and blood pressure as well as in the protection of the renal parenchyma. The main classes of K+ channels (calcium activated (KCa), inward rectifier (Kir), voltage activated (Kv) and ATP sensitive (KATP)) have been found in the renal vessels. In this review, we summarize results available in the literature and our own studies in the field. We compare the ambiguous in vitro and in vivo results. We discuss the role of single types of K+ channels and the integrated function of several classes. We also deal with the possible role of renal vascular K+ channels in the pathophysiology of hypertension, diabetes mellitus and sepsis.

KV7.4 channels participate in the control of rodent renal vascular resting tone

We tested the hypothesis that KV7 channels contribute to basal renal vascular tone and that they participate in agonist‐induced renal vasoconstriction or vasodilation.

Mechanisms of K+ induced renal vasodilation in normo- and hypertensive rats in vivo

Aim:  We investigated the mechanisms behind K+‐induced renal vasodilation in vivo in normotensive Sprague–Dawley (SD) rats and spontaneously hypertensive rats (SHR).

Contribution of K(+) channels to endothelium-derived hypolarization-induced renal vasodilation in rats in vivo and in vitro.

We investigated the mechanisms behind the endothelial-derived hyperpolarization (EDH)-induced renal vasodilation in vivo and in vitro in rats. We assessed the role of Ca2+-activated K+ channels and whether K+ released from the endothelial cells activates inward rectifier K+ (Kir) channels and/or the Na+/K+-ATPase. Also, involvement of renal myoendothelial gap junctions was evaluated in vitro. Isometric tension in rat renal interlobar arteries was measured using a wire myograph. Renal blood flow was measured in isoflurane anesthetized rats. The EDH response was defined as the ACh-induced vasodilation assessed after inhibition of nitric oxide synthase and cyclooxygenase using L-NAME and indomethacin, respectively. After inhibition of small conductance Ca2+-activated K+ channels (SKCa) and intermediate conductance Ca2+-activated K+ channels (IKCa) (by apamin and TRAM-34, respectively), the EDH response in vitro was strongly attenuated whereas the EDH response in vivo was not significantly reduced. Inhibition of Kir channels and Na+/K+-ATPases (by ouabain and Ba2+, respectively) significantly attenuated renal vasorelaxation in vitro but did not affect the response in vivo. Inhibition of gap junctions in vitro using carbenoxolone or 18α-glycyrrhetinic acid significantly reduced the endothelial-derived hyperpolarization-induced vasorelaxation. We conclude that SKCa and IKCa channels are important for EDH-induced renal vasorelaxation in vitro. Activation of Kir channels and Na+/K+-ATPases plays a significant role in the renal vascular EDH response in vitro but not in vivo. The renal EDH response in vivo is complex and may consist of several overlapping mechanisms some of which remain obscure.