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

Abstract

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. British Journal of Pharmacology (2004) 142, 709–718. doi:10.1038/sj.bjp.0705841

Functional Importance of L- and P/Q-Type Voltage-Gated Calcium Channels in Human Renal Vasculature

“…In agreement, T-type channels have been shown to have their dominant effects in smaller human and rat arterioles compared with larger arteries. 32 Also in rodents, T-type channels are most important for the contraction of smaller blood vessels, 33 suggesting that T-type channels might also be of greater significance in human renal arterioles compared with arteries.…”

T-type Ca2+channels and autoregulation of local blood flow

“…However, specific peptide toxins targeting P/Q-type, N-type and R-type VGCCs did not inhibit high-KCl induced Ca 2C entry in rat mesenteric arterioles, 40 so most likely these HVA channels do not play a role in myogenic tone.…”

“…40,46,48,50,113 In small mesenteric arteries from wild-type mice nifedipine (1 mM) abolished myogenic tone at higher pressures, whereas there was no effect on tone at 40 mmHg. In agematched mice deficient in the Ca V 3.1 T-type channels, myogenic tone was abolished at lower pressures (40-80 mmHg), but at pressures above 80 mmHg the spontaneous myogenic tone was abolished by 1 mM nifedipine.…”

“…15,37,38 The L-type channels are all blocked by dihydropyridines, phenylalkylamines and benzothiazepines with similar potencies; however, some peptide toxins (calciseptine, FS-2) may specifically inhibit Ca V 1.2 channels. 20,39,40 The Ca V 1.2 a 1 -subunit isoforms cloned from human intestinal smooth muscle and the heart were both shown to be inherently mechano-sensitive when expressed in HEK-293 cells. 41 The Ca V 2 family (HVA) encoding the P/Q-type (Ca V 2.1a/b: splice variants of the Cacna1a gene), Ntype (Ca V 2.2) and R-type (Ca V 2.3) currents are primarily expressed and functional in neurons where they participate in neurotransmitter release, action potentials, and local Ca 2C transients.…”

“…The P/Q-type channels seem to be expressed in renal preglomerular arterioles, 42 but none of the P/Q-, N-or R-type channels are expressed and functional in mesenteric-terminal arterioles. 40 The Ca V 3 family (LVA) T-type (Tiny, Transient) channels consisting of small and fast-inactivating Ca V 3.1-3 currents are primarily expressed in the peripheral and central nervous system where they are important for repetitive firing of action potentials (for review, see Perez-Reyes, 2003 35 ). However, Ca V 3.1 and Ca V 3.2 channels are also expressed in the heart where they are involved in pacemaking in the sinoatrial node (Ca V 3.1; 43 ) as well as in ventricular hypertrophy where Ca V 3.1 and Ca V 3.2 channels seem to play opposite roles.…”