Capacitative calcium entry as a pulmonary specific vasoconstrictor mechanism in small muscular arteries of the rat

Abstract

(1) The effect of induction of capacitative Ca2+ entry (CCE) upon tone in small (i.d. 200-500 microm) intrapulmonary (IPA), mesenteric (MA), renal (RA), femoral (FA), and coronary arteries (CA) of the rat was examined. (2) Following incubation of IPA with 100 nm thapsigargin (Thg) in Ca2+-free physiological salt solution (PSS), a sustained contraction was observed upon reintroduction of 1.8 mm Ca2+, which was unaffected by either diltiazem (10 microm) or the reverse mode Na+/Ca2+ antiport inhibitor KB-R7943 (10 microm). An identical protocol failed to elicit contraction in MA, RA, or CA, while a small transient contraction was sometimes observed in FA. (3) The effect of this protocol on the intracellular Ca2+ concentration ([Ca2+]i) was assessed using Fura PE3-loaded IPA, MA, and FA. Reintroduction of Ca2+ into the bath solution following Thg treatment in Ca2+-free PSS caused a large, rapid, and sustained increase in [Ca2+]i in all the three types of artery. (4) 100 nm Thg induced a slowly developing noisy inward current in smooth muscle cells (SMC) isolated from IPA, which was due to an increase in the activity of single channels with a conductance of approximately 30 pS. The current had a reversal potential near 0 mV in normal PSS, and persisted when Ca2+-dependent K+ and Cl- currents were blocked; it was greatly inhibited by 1 microm La3+, 1 microm Gd3+, and the IP3 receptor antagonist 2-APB (75 microm), and by replacement of extracellular cations by NMDG+. (5) In conclusion, depletion of intracellular Ca2+ stores with Thg caused capacitative Ca2+ entry in rat small muscular IPA, MA, and FA. However, a corresponding contraction was observed only in IPA. CCE in IPA was associated with the development of a small La3+- and Gd3+-sensitive current, and an increased Mn2+ quench of Fura PE-3 fluorescence. These results suggest that although CCE occurs in a number of types of small arteries, its coupling to contraction appears to be of particular importance in pulmonary arteries.

Figure 1

Thg induces sustained constriction in rat small isolated IPA. (a) The trace shows a typical response of rat 232 μm IPA to 100 nM Thg (applied in Ca²⁺-free PSS). Upon the reintroduction of Ca²⁺ to the myograph bath, a sustained constriction was observed, which was reversed upon removal of Ca²⁺ from the myograph bath. The response increased with repeated Ca²⁺ exposures, yet was reproducible subsequent to the third exposure. This response was unaffected by the voltage-gated channel antagonist diltiazem (10 μM), but was completely abolished by 1 μM La³⁺. (b) shows the mean response from 10 to 26 IPA.

Figure 2

CCE-induced constriction is not observed in rat small MA, RA, CA, or FA. (a, b, c, d) traces show typical responses to 100- nM Thg and subsequent reintroduction of Ca²⁺ in rat MA (210 μm i.d.), RA (303 μm i.d.), CA (187 μm i.d.), and FA (324 μm i.d.), respectively, to 100 nM Thg. No constriction was ever observed in MA, RA, or CA upon reintroduction of Ca²⁺ to the myograph bath (n=8–12). A small transient contraction was observed in four of the 12 FA examined (see Figure 4).

Figure 3

Contraction after depletion of cellular Ca²⁺ store with Thg (100 nM), caffeine (10 mM), and phenylephrine (PE, 10 μM). (a) The trace shows three responses to PE, administered 90 s after the removal of Ca²⁺ from the solution (with addition of 0.5 mM EGTA), in a rat mesenteric resistance artery. After a typical transient control PE contraction (left), stores were refilled by returning Ca²⁺ to the solution, and Thg was applied. This almost abolished the subsequent response to PE in Ca²⁺-free solution. After the application and removal of caffeine and then PE in the absence of Ca²⁺ to ensure further Ca²⁺ store depletion, readdition of Ca²⁺ to the solution (at the end of the long open bar) did not cause contraction. (b) A protocol identical to that described above was carried out in a rat IPA. Here, Ca²⁺ reapplication after store depletion caused a large contraction (right). (c) The mean±s.e.m. amplitude of the three PE contractions were recorded as described above in small FA, MA, RA, and CA. n=4; *P<0.05; **P<0.01.

Figure 4

Simultaneous recording of tension development and [Ca²⁺]_i during application of KPSS and Thg in IPA (upper traces), MA (middle traces), and FA (lower traces). Following addition of KPSS, arteries were exposed to a nominally Ca²⁺-free solution for 10 min. Subsequent reapplication of Ca²⁺ caused a small transient increase in [Ca²⁺]_i. Arteries were then exposed to Ca²⁺-free solution, and 100 nM Thg was applied. Reapplication of Ca²⁺ in the presence of Thg caused a large and sustained increase in [Ca²⁺]_i, which was stable when the cycle of Ca²⁺ removal and replacement was repeated. This increases in [Ca²⁺]_i was associated with a sustained contraction in IPA, with no contraction in MA. A transient contraction was sometimes observed in FA, but tended to wane with further cycles of Ca²⁺ removal and replacement.

Figure 5

Summary of CCE-related tension development and [Ca²⁺]_i in IPA, MA, and FA. Effects of reapplication of extracellular Ca²⁺ following Thg treatment on tension development and [Ca²⁺]_i obtained in individual IPA (n=5), MA (n=6), and FA (n=4) during experiments similar to those shown in Figure 4. The mean±s.e.m. of these responses are shown, with the solid bars representing tension (left axis) and the open bars representing the increase in [Ca²⁺]_i, expressed in terms of the raw R_340/380 values (right axis).

Figure 6

Thg causes an inward current in SMC isolated from rat small IPA. Below, the typical trace of the noisy inward current developing in SMC isolated from small IPA in response to 100 nM Thg. Above, the expanded sections of the same trace showing the dramatic increase of single-channel activity. Holding potential −80 mV, intracellular solution – Cs methanesulphonate, 10 -mM BAPTA. Lines indicate zero current level. Traces shown are typical of five to seven experiments.

Figure 7

Block of Thg-induced inward current. (a) Trivalent cations La³⁺ and Gd³⁺ block Thg-induced inward current; this block could be reversed by chelating lanthanides with EGTA. (b) 2-APB caused partial and reversible block of the Thg-induced inward current, while replacement of all extracellular permeable cations by NMDG⁺ abolished the current completely. (c) Single channels underlying Thg-induced whole-cell current were revealed in the presence of 2-APB, which reduced the opening frequency without affecting conductance. No activity could be observed in NMDG⁺-based solution. Holding potential −80 mV, intracellular solution – Cs methanesulphonate, 10 mM BAPTA. Lines indicate zero current level. Traces shown are typical of six to experiments.

Figure 8

Ion permeability of Thg-induced inward current. (a) Removal of divalent cations with 1 mM EDTA considerably enhanced Thg-induced current. (b) 140 mM K⁺, 140 mM Cs⁺, and 80 mM Ca²⁺ supported the inward current to some extent, although it was smaller than with 140 mM Na⁺ (control). The lines indicate zero current level. Traces shown are typical of six to eight experiments. (c) When the membrane potential was ramped from −100 to +100 mV in the absence of both intra- and extracellular K⁺, Thg treatment (1 μM, 3 min) caused the development of nonrectifying current. (d) After treatment with Thg, the replacement of extracellular Na⁺ for NMDG⁺ suppressed the inward current. Holding potential −80 mV, intracellular solution – Cs methanesulphonate, 10 mM BAPTA. Typical traces are from four to eight experiments.

Figure 9

SMC isolated from IPA possess a Mn²⁺-permeable, La³⁺-, Gd³⁺-, and 2-APB-sensitive basal and Thg-induced influx pathways. (a) Relative rates of basal Fura-PE3 fluorescence quenching with 1 mM Mn²⁺ in unstimulated SMC freshly isolated from IPA. La³⁺ (10 μM, but not 1 μM) caused a significant reduction in the rate of quenching and 10 μM Gd³⁺ suppressed it entirely. Diltiazem (10 μM) had no effect, while 2-APB (75 μM) attenuated the basal quenching. (b) Thg (100 nM) increased the rate of quenching (measured with 0.1 mM Mn²⁺) more than six-fold, and this increase was abolished by 75 μM 2-APB or 1 μM La³⁺. n=10–16, ***P<0.001.