Molecular and Functional Identification of Cyclic AMP-Sensitive BK _Ca Potassium Channels (ZERO Variant) and L-Type Voltage-Dependent Calcium Channels in Single Rat Juxtaglomerular Cells

Abstract

Abstract-This study aimed at identifying the type and functional significance of potassium channels and voltagedependent calcium channels (Ca v ) in single rat JG cells using whole-cell patch clamp. Single JG cells displayed outward rectification at positive membrane potentials and limited net currents between Ϫ60 and Ϫ10 mV. Blockade of K ϩ channels with TEA inhibited 83% of the current at ϩ105 mV. Inhibition of K V channels with 4-AP inhibited 21% of the current. Blockade of calcium-sensitive voltage-gated K ϩ channels (BK Ca ) with charybdotoxin or iberiotoxin inhibited 89% and 82% of the current, respectively. Double immunofluorescence confirmed the presence of BK Ca and renin in the same cell. cAMP increased the outward current by 1.6-fold, and this was inhibited by 74% with iberiotoxin. Expression of the cAMP-sensitive splice variant (ZERO) of BK Ca was confirmed in single-sampled JG cells by RT-PCR. The resting membrane potential of JG cells was Ϫ32 mV and activation of BK Ca with cAMP hyperpolarized cells on average 16 mV, and inhibition with TEA depolarized cells by 17 mV. The cells displayed typical high-voltage activated calcium currents sensitive to the L-type Ca v blocker calciseptine. RT-PCR analysis and double-immunofluorescence labeling showed coexpression of renin and L-type Ca v 1.2. The cAMP-mediated increase in exocytosis (measured as membrane capacitance) was inhibited by depolarization to ϩ10 mV, and this inhibitory effect was blocked with calciseptine, whereas K ϩ -blockers had no effect.

Hypotonicity-Induced Renin Exocytosis from Juxtaglomerular Cells Requires Aquaporin-1 and Cyclooxygenase-2

“…PGE 2 and PGI 2 are likely candidates that activate cAMP-coupled EP2, EP4, and IP receptors on JG cells and enhance potassium current carried through the cAMP-sensitive "zero" variant of the calcium-sensitive voltage-activated potassium (BK Ca ) channels. 11,12 The data presented here show that cAMP/PKA is crucial for transduction of the hypotonicity-induced renin secretory and current response. In agreement with this finding, renin release after a hypotonic challenge is not additive to the response elicited by intracellular application of cAMP.…”

“…Depolarization of the JG cell membrane potential activates voltage-gated calcium channels and calcium influx, which inhibits cAMP-mediated exocytosis of renin. 12 We have shown that hyperpolarization indirectly supports exocytosis of renin by stabilizing membrane voltage in a range far from threshold for calcium channel activation and thus inhibition of renin release. 12 The series of events between a hypotonic challenge and COX-2 activity in JG cells is likely to include cell swelling and PLA 2 activation as observed in several cell types.…”

“…12 We have shown that hyperpolarization indirectly supports exocytosis of renin by stabilizing membrane voltage in a range far from threshold for calcium channel activation and thus inhibition of renin release. 12 The series of events between a hypotonic challenge and COX-2 activity in JG cells is likely to include cell swelling and PLA 2 activation as observed in several cell types. 9 Hypotonicity-induced stimulation of COX-2 activity in cultured cells has been observed previously.…”

Decreased Intracellular Calcium Stimulates Renin Release via Calcium-Inhibitable Adenylyl Cyclase

“…Nonspecific binding was blocked with TBST and 5% BSA for 30 minutes. The cells were washed and then incubated for 1 hour with a primary antibody against mouse renin 18 (Swant, Bellinzona, Switzerland), diluted 1:50 in 5% BSA in TBST. Cells were washed and incubated with a goat anti-mouse secondary antibody labeled with Alexa Fluor 488 green fluorescent dye (Alexa Fluor, Invitrogen) diluted 1:100 in 5% BSA in TBST for 1 hour.…”

Cyclic AMP increases cytoplasmic free calcium in renin-secreting cells from rat kidney

“…In addition, stimulation by isoproterenol + IBMX left membrane potential unaltered at approx. -60 mV (see also Bührle et al 1986) but is at variance with the hyperpolarisation observed by Fishman (1976) and Friis et al (2003).…”