Abstract
The purpose of our study was to determine whether Gi-mediated control over adenylyl cyclase in preglomerular arteriolar smooth muscle cells (PGASMC) is enhanced in the spontaneously hypertensive rat (SHR). PGASMC were cultured from preglomerular microvessels isolated from adult SHR (14-15 wk of age) and age-matched WKY rats. Confluent monolayers of cells in third passage were used for the experiments. cAMP released into the media (30 min) as well as cellular levels of cAMP were measured in the presence of a phosphodiesterase inhibitor, 1-isobutyl-3-methyl-xanthine (IBMX; 100 μM) and expressed as pmol/mg protein. Total (released + cellular) cAMP was significantly lower in SHR (14.19 ± 2.30 pmol/mg protein) as compared with WKY (28.3 ± 3.04 pmol/mg protein). Correspondingly, the released (4.6 ± 0.4 pmol/mg protein) as well as cellular (9.78 ± 2.18 pmol/mg protein) cAMP levels were also significantly lower in SHR when compared with WKY (8.85 ± 1.26 and 18.86 ± 2.0 pmol/mg protein, respectively). The steady-state levels of none of the Giα subunits, namely Giα1, Giα2 and Giα3, were higher in the SHR PGASMC. Pertussis toxin treatment (PTX; 100 ng/ml; 24 hr) caused complete ADP-ribosylation of Giα subunits in both WKY and SHR PGASMC. The same treatment of PTX also produced a significant increase in total cAMP in SHR, but not in WKY, such that the total cAMP levels after PTX treatment were not significantly different between the two strains. Interestingly, PTX significantly increased the released (20.26 ± 0.90 pmol/mg protein) but not the cellular (13.63 ± 1.63 pmol/mg protein) cAMP in SHR. Forskolin (1 μM) induced similar increases in total cAMP and isoproterenol (1 μM) caused greater increases in total cAMP in SHR cells compared with WKY cells. These data strongly suggest that in SHR PGASMC total adenylyl cyclase activity is not altered. Furthermore, steady-state expressions of G iα-1, Giα-2 and Giα-3 are not increased whereas Gi -mediated inhibition of adenylyl cyclase is augmented in SHR PGASMC.
Footnotes
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Send reprint requests to: Dr. Subhash J. Vyas, Center for Clinical Pharmacology, Scaife Hall, Room 623, University of Pittsburgh Medical Center, 200 Lothrop Street, Pittsburgh, PA 15213-2582.
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↵1 This work was supported by Grants HL-35909 and HL-55314 from the National Institutes of Health Bethesda, MD. A portion of this work was presented at the Experimental Biology, 1996 Annual Meeting, Washington, D.C., April 14-17 and was published as an abstract (FASEB J10:A698, 1996).
- Abbreviations:
- SHR
- spontaneously hypertensive rat
- WKY
- Wistar-Kyoto
- cAMP
- adenosine 3′, 5′-cyclic monophosphate
- Ang II
- angiotensin II
- ISO
- isoproterenol
- Gi, guanine nucleotide-binding inhibitory protein
- Gs,guanine nucleotide-binding stimulatory protein
- IBMX
- 3-isobutyl-1-methyl-xanthine
- PTX
- pertussis toxin
- PGASMC
- preglomerular arteriolar smooth muscle cells
- DMEM
- Dulbecco’s modified Eagle medium
- PBS
- phosphate-buffered saline
- Received March 28, 1997.
- Accepted January 20, 1998.
- The American Society for Pharmacology and Experimental Therapeutics
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Guanine Nucleotide-Binding Inhibitory Protein-Mediated Inhibition of Adenylyl Cyclase Is Enhanced in Spontaneously Hypertensive Rat Preglomerular Arteriolar Smooth Muscle Cells
