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

Abstract

1. In this study, intracellular Ca(2+) was measured as the Fura-2 ratio (R) of fluorescence excited at 340 and 380 nm (F(340)/F(380)) in nonpressurized rat mesenteric small arterioles ( (lumen diameter) 10-25 microm). 2. The response to depolarization using 75 mm KCl was an increase in R from a baseline of 0.96+/-0.01 ([Ca(2+)](i) approximately 74 nm) to 1.04+/-0.01 ( approximately 128 nm) (n=80). The response to 75 mm K(+) was reversibly abolished in Ca(2+)-free physiological saline solution, whereas phentolamine (10 microm) or tetrodotoxin (1 microm) had no effects. LaCl(3) (200 microm) inhibited 61+/-9% of the response. 3. A [K(+)]-response curve indicated that the Ca(2+) response was activated between 15 and 25 mm K(+). The data suggest that the Ca(2+) response was caused by the activation of voltage-dependent Ca(2+) channels. 4. Mibefradil use dependently inhibited the Ca(2+) response to 75 mm K(+) by 29+/-2% (100 nm), 73+/-7% (1 microm) or 89+/-7% (10 microm). Pimozide (500 nm) use dependently inhibited the Ca(2+) response by 85+/-1%. 5. Nifedipine (1 microm) inhibited the Ca(2+) response to 75 mm K(+) by 41+/-12%. The response was not inhibited by calciseptine (500 nm), omega-agatoxin IVA (100 nm), omega-conotoxin MVIIA (500 nm), or SNX-482 (100 nm). 6. Using reverse transcriptase-polymerase chain reaction, it was shown that neither Ca(V)2.1a (P-type) nor Ca(V)2.1b (Q-type) voltage-dependent Ca(2+) channels were expressed in mesenteric arterioles, whereas the Ca(V)3.1 (T-type) channel was expressed. Furthermore, no amplification products were detected when using specific primers for the beta(1b), beta(2), or beta(3) auxiliary subunits of high-voltage-activated Ca(2+) channels. 7. The results suggest that the voltage-dependent Ca(2+) 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 Ca(2+) influx observed was the result of a T-type window current is discussed.

Figure 1

Micrograph of Fura-2-loaded arteriole in isolated rat mesentery. The tissue was excited at 340 nm and emission collected at 510 nm, giving rise to the bright fluorescence seen in the arteriolar wall. For the purpose of illustration, a dimmed transmission light was used to distinguish the arteriolar lumen and wall from the mesenteric tissue. The few bright spots seen in the mesentery probably correspond to fibroblasts loaded with Fura-2. The freshly isolated tissue was glued to a cover slide and subsequently loaded with Fura-2/AM in the recording chamber. In some cases, excess mesenteric tissue was carefully removed by microdissection prior to Fura loading and arterioles mounted between two suction pipettes during recordings.

Figure 2

(a) Original recording of Fura-2 ratio (F₃₄₀/F₃₈₀) in mesenteric arteriole. Sustained depolarization induced by 75 mM K⁺ (K) resulted in an increase of intracellular [Ca²⁺] (F₃₄₀/F₃₈₀) from the baseline level. The Ca²⁺ response was reversibly abolished in the presence of Ca²⁺-free PSS with Ca²⁺ chelator (EGTA). (b) Summary of Ca²⁺ responses to 75 mM K⁺ shown as % of baseline Fura-2 ratio. Compared to the control stimulation (K75 Control), the Ca²⁺ response was not altered by treatment with phentolamine (10 μM) or addition of TTX (1 μM) on top of phentolamine. However, the response was partially blocked in the presence of 200 μM La³⁺. The number of arterioles used is shown within parenthesis. ^*P<0.05, ^**P<0.01; t-test vs control stimulation.

Figure 3

In a number of unpaired experiments, arterioles were subjected to depolarization with varying extracellular [K⁺]. The concentration−peak response curve indicates activation of Ca²⁺ entry between 15 and 25 mM extracellular K⁺ and the response is maximal at approximately 75 mM K⁺. The number of arterioles used is shown within parenthesis.

Figure 4

Effects of various Ca²⁺ channel blockers shown as % inhibition of the Ca²⁺ response to 75 mM K⁺. The L-type Ca²⁺ channel blocker nifedipine (1 μM) inhibited 40% of the response, whereas there was no effect of a highly specific L-type blocker, the neurotoxin calciseptine (500 nM). Highly specific neurotoxin blockers of P/Q-type (ω-agatoxin IVA, 100 nM), N-type (ω-conotoxin MVIIA, 500 nM), or R-type Ca²⁺ channels (SNX-482, 100 nM) did not inhibit the response to high K⁺. There was a minor increase of the Ca²⁺ response in the presence of ω-agatoxin IVA. The number of arterioles is within parenthesis. ^*P<0.05; t-test vs untreated control experiments.

Figure 5

Effects of the T-type Ca²⁺ channel blockers mibefradil and pimozide. (a) In this original trace, an arteriole was stimulated repeatedly (12 stimulations of 40 s duration) with 75 mM K⁺ (K). After four stimulations mibefradil (1 μM) was applied for a total period of ∼10 min. The inhibition induced by mibefradil was maximal after three to four stimulations, and this time-independent effect is referred to as use-dependent block. (b) Open columns depict % inhibition of the first stimulation (initial block) and hatched columns depict inhibition of the last stimulation (use-dependent block) in the presence of mibefradil at all concentrations tested (n=4−5). The untreated control experiments are shown here as 0.0 μM mibefradil (n=4). (c) Ca²⁺ responses to 75 mM K⁺ (K) in an arteriole exposed to 500 nMof the antipsychotic pimozide, clearly showing a use-dependent effect of the drug. A small reversible decrease of the baseline ratio is seen after the addition of pimozide. (d) Summary of responses obtained in arterioles exposed to pimozide (n=4) and in vehicle control experiments (n=4). ^*P<0.05, ^**P<0.01, and ^*** P<0.001; t-test vs untreated or vehicle control experiments.

Figure 6

Agarose-gel electrophoresis showing PCR products obtained when using specific primers for selected α₁- and β-subunits of voltage-dependent Ca²⁺ channels and cDNA from mesenteric arterioles. The Ca_V2.1-P and Ca_V2.1-Q are alternative splice variants of the same gene and corresponds to the P-type and Q-type α₁-subunits, respectively. Ca_V3.1-T corresponds to the α_1G-subunit of T-type channels. β-2 (cardiac), β-3 (smooth muscle), and β-B (β_1b, neuronal) are known β-subunits of high-voltage-activated Ca²⁺ channels. Specific primer for β-actin was also included in the reactions. Pooled cDNA from mesenteric arterioles in each of two rats were used (Prep 1, Prep 2). Appropriate controls are shown in which cDNA was omitted (neg. control) or 50 ng template cDNA isolated from various rat organs (cerebral cortex, left cardiac ventricle, or renal inner medulla) was included in the PCR (pos. control).