Immunoblot and immunohistochemical comparison of murine monoclonal antibodies specific for the rat D1a and D1b dopamine receptor subtypes.

The two D1-like dopamine receptor subtypes, D1a and D1b, are structurally similar and pharmacologically indistinguishable using currently available ligands. To differentiate between the D1-like dopamine receptor subtypes, murine monoclonal antibodies to the rat Dla and the rat D1b dopamine receptor have been prepared. Rat D1-like and D2-like dopamine receptors expressed in Sf9 cells were used to verify the immunospecificity of the monoclonal anti-(D1a dopamine receptor) and anti-(D1b dopamine receptor) antibodies using immunoblot and immunohistochemical techniques. These two antibodies were used to compare the temporal dynamics of D1-like dopamine receptors expressed in Sf9 cells following infection with recombinant baculovirus and to monitor the partial purification of detergent solubilized receptors following ion exchange chromatography. Immunoreactivity of the anti-(D1a receptor) antibody was observed in the striatum and cortical regions of the rat brain using immunoblot techniques. No reactivity on immunoblots was observed for the anti-(D1b receptor) antibody using rat brain tissue, probably due to the low levels of receptor expression. For immunohistochemical studies using rat brain slices, the anti-(D1a receptor) antibody heterogeneously labeled cells and punctate processes within the striatal neuropil while labeling in the adjacent cerebral cortex was weak. Anti-(D1b receptor) antibody immunoreactivity was weak in the .striatum and generally limited to sparse perikarya in the dorsal region. However, immunoreactivity was observed in numerous cells within the vertical and horizontal limbs of the diagonal band and in the ventral pallidum. Immunoreactivity of the anti-(D1b receptor) antibody was also observed in layer V pyramidal neurons of the frontal sensorimotor cortex.

Genetic polymorphisms at the rat and murine loci coding for dopamine D2-like receptors.

Southern blot hybridization techniques have been used to identify genetic polymorphisms at the D2, D3 and D4 dopamine receptor loci in mice and rats. Genomic DNA from a panel of outbred and inbred strains of rats and inbred strains of mice was digested with a variety of restriction endonucleases. After separation of the restriction digests on the basis of size using agarose gel electrophoresis, 32P-labeled DNA probes coding for the rat D2, D3 and D4 dopamine receptors were used to identify a series of genetic polymorphisms at each of these receptor loci. Genetic polymorphisms were found for the rat and murine D2, D3 and D4 dopamine receptor loci. It is anticipated that these genetic polymorphisms will be useful in pharmacogenetic studies to determine the influence of the D2-like receptors in reward and addictive behaviors.

Molecular characterization of a mammalian smooth muscle myosin light chain kinase.

A 5.6-kilobase cDNA clone has been isolated which includes the entire coding region for the myosin light chain kinase from rabbit uterine tissue. This cDNA, expressed in COS cells, encodes a Ca2+/calmodulin-dependent protein kinase with catalytic properties similar to other purified smooth muscle myosin light chain kinases. A module (TLKPVGNIKPAE), repeated sequentially 15 times, has been identified near the N terminus of this smooth muscle kinase. It is not present in chicken gizzard or rabbit skeletal muscle myosin light chain kinases. This repeat module and a subrepeat (K P A/V) are similar in amino acid content to repeated motifs present in other proteins, some of which have been shown to associate with chromatin structures. Immunoblot analysis after sodium dodecyl sulfate-polyacrylamide gel electrophoresis, used to compare myosin light chain kinase present in rabbit, bovine, and chicken smooth and nonmuscle tissues, showed that within each species both tissue types have myosin light chain kinases with indistinguishable molecular masses. These data suggest that myosin light chain kinases present in smooth and nonmuscle tissues are the same protein.

Structural changes during the early onset of experimental hypertension: trophic influences of the renin-angiotensin system.

All forms of established hypertension are characterised by hypertrophy of the heart and blood vessels. Although these structural alterations are a normal adaptation to raised blood pressure, it has been suggested that hypertrophy may develop by a mechanism independent of pressure. Some investigators claim that in the young spontaneously hypertensive rat, hypertrophy of the blood vessels and heart is present at a time before the rise in pressure. Evidence also exists to support the role of angiotensin II in directly influencing growth within the vasculature. The present study confirms that hypertrophy of the mesenteric resistance vessels is present in the 3-week old spontaneously hypertensive rat. However, enhanced vascular structure was also associated with a higher mean arterial blood pressure. No increase in activity of the renin-angiotensin system could be detected. These observations raise doubts as to whether hypertrophy of the blood vessels can develop independently of raised pressure.

Angiotensin II causes vascular hypertrophy in part by a non-pressor mechanism.

Angiotensin II, when given in low doses, raises blood pressure slowly. When tested in vitro on vascular smooth muscle cells, it has mitogenic and trophic effects; it is not known if it has these effects in vivo. Our purpose was to determine whether vascular hypertrophy develops during slow pressor infusion of angiotensin II and, if so, whether it is pressure induced. Three experiments were done in rats infused subcutaneously with angiotensin II (200 ng/kg/min) by minipump for 10-12 days. Experiment 1: Angiotensin II gradually raised systolic blood pressure (measured in the tail) from 143 +/- 2 to 208 +/- 8 mm Hg (mean +/- SEM), significantly suppressing plasma renin and increasing threefold (NS) plasma angiotensin II. There was no loss of peptide in the pump infusate when tested at the end of the experiment. Experiment 2: In the perfused mesenteric circulation, vasoconstrictor responses to norepinephrine, vasopressin, and KCl were enhanced in rats given a slow pressor infusion of angiotensin II, but sensitivity of responses was not altered. This combination of changes suggests that vascular hypertrophy develops during slow pressor infusion of angiotensin II. Experiment 3: Vessel myography was done after angiotensin II infusion with and without a pressor response. Angiotensin II raised systolic blood pressure, increased heart weight, and produced myographic changes of vascular hypertrophy in the mesenteric circulation, increasing media width, media cross-sectional area, and media/lumen ratio. Hydralazine given with angiotensin II prevented the rise of pressure and the cardiac effect but not the vascular changes. Two-way analysis of variance showed that angiotensin II significantly increased media width, media cross-sectional area, and media/lumen ratio, all independent of hydralazine. Thus, although hydralazine inhibits the pressor and cardiac effects of angiotensin II, suggesting a pressor mechanism for the cardiac change, it does not inhibit structural vascular change, which suggests that at least part of the effect has a non-pressor mechanism.

Brief angiotensin converting enzyme inhibitor treatment in young spontaneously hypertensive rats reduces blood pressure long-term.

Our study examines the long-term cardiovascular effects after a brief period of angiotensin converting enzyme (ACE) inhibitor treatment in young spontaneously hypertensive rats (SHR). SHR were treated with perindopril (3 mg/kg/day) by gavage from 2 to 6, from 6 to 10, or from 2 to 10 weeks of age. Systolic blood pressure was measured in the tail weekly until 25 weeks of age. Corresponding control groups received distilled water for the same periods. In each treatment group blood pressure was reduced significantly during treatment, rose when treatment stopped, but plateaued significantly below control SHR thereafter. This difference in blood pressure at 25 weeks of age was due to reduced total peripheral resistance as determined by microsphere methods, but plasma renin activity and angiotensin II concentrations were not different. Cardiac hypertrophy was also reduced in treated SHR. In a separate experiment, perindopril treatment from 6 to 10 weeks of age resulted in a significant reduction in the media/lumen ratios of mesenteric resistance vessels at 32 weeks of age. Concomitant administration of angiotensin II with perindopril from 6 to 10 weeks of age not only prevented the long-term effects on blood pressure seen with perindopril treatment alone but was associated with cardiovascular hypertrophy in excess of untreated control SHR. Finally, perindopril given for a shorter period (6 to 7 weeks) or later in life (20 to 24 weeks) had no significant long-term effects on blood pressure. These results demonstrate that a 4-week period of ACE inhibitor treatment in young SHR is sufficient to prevent the full expression of genetic hypertension and cardiovascular hypertrophy and that angiotensin II might be important in the development of hypertension in this model, its role in later life being less important.

Vascular hypertrophy, renin and blood pressure in the young spontaneously hypertensive rat.

1. Cardiovascular reactivity, blood vessel morphology, blood pressure and the activity of the renin-angiotensin system were determined in the 3-week-old spontaneously hypertensive (SHR), Wistar-Kyoto (WKY) and outbred Wistar (WIS) rat. 2. In an isolated perfused mesenteric artery preparation the SHR had a significantly increased maximum response to KCl and noradrenaline (P less than 0.02) compared with the WKY. Using a myograph, vascular structure was measured over a range of resistance arteries and showed a significant correlation between lumen diameter and both media cross-sectional area and thickness, with the regression line for the SHR shifted upwards indicating both increased media area and thickness. This was associated with a slight, but significant, narrowing of the lumen (P less than 0.01) and an increased media/lumen ratio (0.049 +/- 0.01, 0.034 +/- 0.007, 0.036 +/- 0.008 for SHR, WKY and WIS, respectively, means +/- SD P less than 0.001). The SHR had a greater heart/body weight ratio than either the WKY or the WIS (P less than 0.001). 3. Both mesenteric artery and membrane protein content were higher in the SHR, indicating an increase in cell size or number. 4. Plasma renin activity (means +/- SD) was lower in the SHR (1.0 +/- 0.7 pmol of angiotensin I h-1 ml-1) than in the WKY (2.2 +/- 1.2 pmol of angiotensin I h-1 ml-1, P less than 0.001) but not different from that in the WIS (1.2 +/- 0.8 pmol of angiotensin I h-1 ml-1). Mesenteric artery vascular renin concentration was also lower in the SHR (P = 0.06).(ABSTRACT TRUNCATED AT 250 WORDS)