Purinoceptor-Mediated Calcium Signaling in Preglomerular Smooth Muscle Cells

Abstract

Abstract-The current studies were performed to determine the contribution of calcium mobilization and voltage-dependent calcium influx to the increase in [Ca 2ϩ ] i elicited by ATP and UTP. Suspensions of freshly isolated smooth muscle cells were prepared from preglomerular microvessels by enzymatic digestion and loaded with the Ca 2ϩ -sensitive dye fura 2. ] i response to ATP and UTP was also assessed in the absence of extracellular calcium. In these studies, exposure to 100 mol/L ATP induced a transient peak increase in [Ca 2ϩ ] i , with the plateau phase being totally abolished. In contrast, exposure to 100 mol/L UTP under calcium-free conditions resulted in no detectable change in the UTP-mediated increase in [Ca 2ϩ ] i . The role of L-type calcium channels in the response was assessed with the calcium channel antagonist diltiazem. Incubation with diltiazem (10 mol/L) markedly reduced the response to ATP, whereas the response to UTP was only slightly reduced. These data demonstrate that both ATP and UTP directly stimulate a biphasic increase in [Ca 2ϩ ] i in renal microvascular smooth muscle cells. Furthermore, the data suggest that the elevation of [Ca 2ϩ ] i elicited by ATP is largely dependent on calcium influx through L-type calcium channels, whereas the response to UTP appears to derive primarily from mobilization of calcium from intracellular stores. The mechanism of this vasoconstriction is at least partially dependent on the activation of voltage-gated calcium channels. 1 Subsequently, it was shown that P2 receptor inactivation or blockade significantly attenuated pressure-mediated afferent arteriolar vasoconstrictor responses. 5 This observation led to the postulate that activation of P2 receptors plays an important role in mediating renal microvascular autoregulatory responses; however, the specific P2 receptor subtype involved remains to be determined.Recently, it was reported that P2 receptor-mediated renal vasoconstriction can be elicited by several different purine-or pyrimidine-based compounds that exhibit varying selectivity for different P2 receptor subtypes. 5,6 ATP and UTP evoke strikingly different afferent arteriolar vasoconstrictor responses; thus, suggesting the involvement of P2X (ATP) and P2Y (UTP) purinoceptor subtypes. 5 P2X and P2Y purinoceptors are structurally different receptor proteins that use different signal transduction pathways 7-13 ; however, the signal transduction cascade for each has not been thoroughly investigated in the renal microcirculation. Clarification of the calcium signaling pathways accessed by ATP and UTP is important to advancing our understanding of the mechanisms by which P2 receptors influence renal microvascular tone and may provide insight into the specific P2 receptor subtype or subtypes involved in the autoregulatory response.We have recently established the methods needed for obtaining viable vascular smooth muscle cells from freshly isolated preglomerular microvascular tissue. 14 We used this preparation to determine the effect of ATP a...

Physiological role for P2X1 receptors in renal microvascular autoregulatory behavior

“…Functional studies from our laboratory (7,19,20,23) and others (16,46) have established that afferent arterioles express P2X and P2Y receptors. While full characterization of the P2 receptor complement has not been completed, evidence strongly supports the presence of P2X 1 and P2Y 2 receptors on the preglomerular vascular smooth muscle (7, 19-23, 47, 48).…”

“…Studies suggest that P2 receptors contribute to the regulation of renal microvascular function (1,(7)(8)(9)(10)(16)(17)(18). P2X and P2Y receptors are expressed by preglomerular microvascular smooth muscle (7,8,16,(19)(20)(21)(22)(23), and previous studies implicate P2 receptors in mediating autoregulatory behavior (7-9, 17, 24). Unfortunately, due to the lack of subtype-specific P2 receptor antagonists, the specific P2 receptor subtype involved in the autoregulatory response has eluded identification.…”

Cytochrome P450 and Cyclooxygenase Metabolites Contribute to the Endothelin-1 Afferent Arteriolar Vasoconstrictor and Calcium Responses

“…The cortical tissue was pressed through a sieve (180-m mesh), and the sieve retentate was washed several times with ice-cold low-calcium PSS and enzymatically digested to obtain renal microvascular smooth muscle cells as previously described. 20 The dispersed renal microvascular smooth muscle cells were gently resuspended in 1.0 mL Dulbecco's minimum essential medium supplemented with 20% FCS, 100 U/mL penicillin, and 200 g/mL streptomycin. Renal microvascular cell suspensions were stored on ice until use.…”

“…The fluorescence data were calibrated as previously described. 20 Measurement of [Ca 2ϩ ] i in single microvascular smooth muscle cells was performed as previously described. 20 …”

Impaired Ca 2+ Signaling Attenuates P2X Receptor–Mediated Vasoconstriction of Afferent Arterioles in Angiotensin II Hypertension

“…14 -18 ATP induces vasoconstriction by activating P2 receptors on preglomerular microvascular smooth muscle cells. 19,20 This vasoconstriction involves activation of P2X and P2Y receptors. It has been established that purinoceptors, especially P2X 1 receptors, play a critical role in mediating pressure-dependent autoregulatory adjustments in afferent arteriolar diameter.…”