Introduction
Three main cellular messengers are involved in the intracellular control of renin secretion. Calcium is an inhibitory second messenger and is increased after exposure of the juxtaglomerular cells (JG cells) to vasocontrictors. Cyclic AMP stimulates the secretory process and is increased after exposure to vasodilator hormones. The role of cGMP is less clear. It was recently suggested, however, that the overall effect of intracellular cGMP on renin secretion is due to its inhibition of the cAMP-specific phosphodiesterase type IIIa (PDE IIIa) (Kurtz & Wagner, 1999).
Methods
The patch clamp technique was applied to isolated single rat JG cells in order to measure membrane capacitance (Cm), which is directly proportional to membrane surface area, and hence a direct measure of exocytosis/endocytosis (Neher & Marty, 1982). Cm was measured with the "sine + dc" method using the LockIn extension of the EPC-9 PULSE software. Cm was measured for 10 min in the tight-seal whole cell configuration.
Results
All cells in this study (148 cells) displayed an I-V curve that was similar to previously published data from JG cells in afferent arterioles (Kurtz & Penner, 1989): outward rectification at positive membrane potentials and very limited net currents between –30 to 0 mV. The JG cells had an average Cm value of 2.88 ± 0.06 pF (mean ± SEM, n=148), which is equivalent to a cell surface area of 288 µm2, a diameter of 10 µm, and a volume of 463 µm3, assuming that the cells are spherical and that the specific capacitance is 1 µF/cm2. During the recording period (10 min), basal Cm did not change significantly (3.8 ± 1.8%, n=6, P>0.05). Cyclic AMP (1 µM), added to the cell cytosol via the patch pipette, increased Cm significantly (15.3 ± 5.2%, n=7), whereas higher concentrations of cAMP (10 and 100 µM) activated a membrane retrieval response (% change in Cm were -4.1 ± 1.8% (n=4) and -10.7 ± 8.6% (n=4), respectively). The PDE IIIa inhibitor trequinsin (200 nM) mimicked the effect of high cAMP (Cm decreased 17.4 ± 6%, n=5). The protein kinase A inhibitor Rp-cAMPS (25 µM) completely blocked the effect of trequinsin and of cAMP (1 µM) (% change in Cm were 0.5 ± 0.75% (n=4) and –1.8 ± 1.6% (n=4), respectively). Cyclic GMP mimicked the effects of cAMP on Cm at a 10 fold higher concentration. Thus, cGMP (10 µM) resulted in an increase in Cm amounting to 4.4 ± 1.5% (n=4), whereas cGMP (100 µM) decreased Cm by 5 ± 0.7% (n=4). The effect of intracellular cGMP was blocked by Rp-cAMPS.
Conclusions
At low cAMP concentrations a net increase in Cm predominates, whereas at high concentrations, in which exocytosis is also likely to be strongly accelerated, membrane retrieval becomes significant and determines the net change in Cm. Cyclic GMP mimicked the effects of cAMP, which was to be expected of an endogenous inhibitor of the cAMP-specific PDE IIIa. Thus, cGMP increases cAMP concentration by inhibition of its degradation by PDE IIIa.
References
Kurtz, A. & Wagner, C. 1999. J Exp Biol 202, 219-225
Neher, E. & Marty, A. 1982. Proc Natl Acad Sci USA 79, 525-535
Kurtz, A. & Penner, R. 1989. Proc Natl Acad Sci USA 86, 3423-3427