Introduction
Of six types of voltage-dependent calcium channels (VDCC), only L- and T-type channels have been identified in renal vascular smooth muscle cells. mRNA of the alpha 1A subunit encoding the neuronal P/Q-type calcium channel has previously been demonstrated in kidney cortex (Yu et al. 1992). We decided to investigate whether this channel was expressed in smooth muscle cells and contributed functionally to contraction in renal resistance vessels.
Methods
The expression of the a1A subunit mRNA was investigated with RT-PCR analysis and Southern Blotting in microdissected rat pre- and postglomerular vessels, in cultures of smooth muscle cells from rat preglomerular vessels, and in cultured rat mesangial cells. With Western blotting using a commercial antibody raised against the highly purified peptide CNA1, corresponding to residues 865-881 of alpha 1A subunit of rat brain VDCC the expression of the alpha 1A subunit protein was investigated in aorta, in cultured aortic smooth muscle cells (A7r5), cultured smooth muscle cells from preglomerular vessels and cultured mesangial cells. Immunolabeling for the alpha 1A subunit and renin was tested in microdissected HCl-treated renal resistance vessels. The secondary antibody was Horse Radish Peroxidase-conjugated goat anti-rabbit IgG and the vessels were incubated with a diaminobenzidin (DAB) staining. Rabbit afferent arterioles were microdissected and perfused to test whether P/Q-type VDCC are involved in excitation contraction coupling using w-agatoxin-IVA a blocker of P/Q-type VDCC. At concentrations less than 10 nM the toxin is specific for the P-type VDCC.
Results
P/Q-type, alpha 1A mRNA expression was identified in microdissected rat preglomerular vessels, in cultured smooth muscle cells from rat preglomerular vessels, in mesangial and A7R5 aortic smooth muscle cells. RT-PCR analysis for the a1A subunit on microdissected postglomerular vessels was negative. In immunoblots, aorta, brain as well as A7r5 cells, cultured renal smooth muscle cells and cultured mesangial cells gave rise to bands of the expected size (190 kD) when labeled with the anti-a1A antibody. Immunolabeling of HCl maceration-microdissected renal vascular segments with the anti-a1A antibody showed expression of the protein throughout the preglomerular vasculature, as well as in the glomeruli. Using microperfusion we found that w-agatoxin-IVA totally blocked the K+ induced contraction in all specimens at 10-14 M. The blockade was reversible.
Conclusions
We conclude that alpha 1A subunit mRNA and protein (encoding P/Q type VDCC) are expressed in smooth muscle cells from renal preglomerular resistance vessels and aorta, as well as mesangial cells, and that P-type VDCC contribute functionally to depolarization-mediated contraction cells of renal afferent arterioles.
References
Yu, A. S., S. C. Hebert, B. M. Brenner, and J. Lytton. 1992. Proc. Natl. Acad. Sc.i USA 89: 10494-10498.