Conrado Johns

Models of Experimental Hypertension in Mice

Experimental models of hypertension in various animals are useful in the research of vasoactive mechanisms. Recombinant DNA technology has produced genetically engineered animals, mostly mice, useful in hypertension research. However, the development of hypertensive models in mice is fraught with technical difficulties. We describe here the successful development in mice of two common types of experimental hypertension: the renovascular two-kidney, one clip and mineralocorticoid deoxycorticosterone-salt models. By adapting technology previously used in rats, we succeeded in developing hypertension (defined as systolic pressures higher than 140 mm Hg) in more than 50% of mice so treated. We also adapted the methodology for indirect tail-cuff blood pressure measurements as well as for direct intra-arterial monitoring of blood pressure in conscious, freely moving mice. Application of these techniques in transgenic or gene knockout mice with altered vasoactive hormones or receptors should allow elucidation of the role of the target gene products in various types of hypertension.

Vasoactive Potential of the B1 Bradykinin Receptor in Normotension and Hypertension

Abstract— The B1 type receptor of bradykinin (Bk B1R) is believed to be physiologically inert but highly inducible by inflammatory mediators and tissue damage. To explore the potential participation of the Bk B1R in blood pressure (BP) regulation, we studied mice with deleted Bk B2R gene with induced experimental hypertension, either salt-dependent (subtotal nephrectomy with 0.5% NaCl as drinking water) or renin/angiotensin-dependent (renovascular 2-kidney–1-clip). Compared with the wild-type controls, the B2R gene knockout mice had a higher baseline BP (109.7±1.1 versus 101.1±1.3 mm Hg, P =0.002), developed salt-induced hypertension faster (in 19.3±2.3 versus 27.7±2.4 days, P =0.024), and had a more severe end point BP (148±3.7 versus 133±3.1 mm Hg, P <0.05). On the contrary, renovascular hypertension developed to the same extent (149.7±4.3 versus 148±3.6 mm Hg) and in the same time frame (14±2.2 versus 14±2.1 days). A bolus infusion of a selective B1R antagonist at baseline produced a significant hypertensive response (by 11.4±2 mm Hg) in the knockout mice only. Injection of graded doses of a selective B1R agonist produced a dose-dependent hypotensive response in the knockout mice only. Assessment of tissue expression of B1R and B2R genes by reverse transcription–polymerase chain reaction techniques revealed significantly higher B1R mRNA levels in the B2R knockout mice at all times (normotensive baseline and hypertensive end points). At the hypertensive end points, there was always an increase in B1R gene expression over the baseline values. This increase was significant in cardiac and renal tissues in all hypertensive wild-type mice but only in the clipped kidney of the renovascular knockout mice. The B2R gene expression in the wild-type mice remained unaffected by experimental manipulations. These results confirm the known vasodilatory and natriuretic function of the Bk B2R; they also indicate that in its absence, the B1R can become upregulated and assume some of the hemodynamic properties of the B2R. Furthermore, they indicate that experimental manipulations to produce hypertension also induce upregulation of the B1R, but not the B2R, in cardiac and renal tissues.

Role of the α2B-Adrenergic Receptor in the Development of Salt-Induced Hypertension

Abstract —Salt sensitivity is a common trait in patients with essential hypertension and seems to have both an inherited and an acquired component (eg, is influenced by aging and renal insufficiency). Experimental evidence suggests that salt loading induces hypertension via a neurogenic mechanism mediated by the α2-adrenergic receptors (α2-AR). To explore the α2-AR subtype involved in this mechanism, we studied 2 groups of mice genetically engineered to be deficient in one of the 3 α2-AR subtype genes (either α2B-AR +/− or α2C-AR −/− knockout mice) compared with their wild-type counterparts. The mice (n=10 to 14 in each group) were submitted to subtotal nephrectomy and given 1% saline as drinking water for up to 35 days. Blood pressure (BP) was monitored by tail-cuff readings and confirmed at the end point by direct intra-arterial BP recording. The α2B-AR–deficient mice had an attenuated BP response in this protocol (baseline 101.8±2.7 versus end point 109.9±2.8 mm Hg), whereas the BP of their wild-type counterparts went from a baseline 101.9±2.3 to an end point 141.4±7.1 mm Hg. The other 2 groups had BP increases of 44.6±5.17 and 46.7±7.01 mm Hg, with no difference between the mice deficient in the α2C-AR gene subtype versus their wild-type counterparts. Body weight, renal remnant weight, and residual renal function were no different among groups. These data suggest that a full complement of α2B-AR genes is necessary to raise BP in response to dietary salt loading, whereas complete absence of the α2C-AR subtype does not preclude salt-induced BP elevation. It is unclear whether the mechanism(s) involved in this process are of central origin (inability to increase sympathetic outflow), vascular origin (inability to vasoconstrict), or renal origin (inability to retain excess salt and fluid).

Sympathoinhibitory Function of the α2A-Adrenergic Receptor Subtype

Abstract —Presynaptic α 2 -adrenergic receptors (α 2 -AR) are distributed throughout the central nervous system and are highly concentrated in the brain stem, where they contribute to neural baroreflex control of blood pressure (BP). To explore the role of the α 2A -AR subtype in this function, we compared BP and plasma norepinephrine and epinephrine levels in genetically engineered mice with deleted α 2A -AR gene to their wild-type controls. At baseline, the α 2A -AR gene knockouts (n=11) versus controls (n=10) had higher systolic BP (123±2.5 versus 115±2.5 mm Hg, P P P 2A -AR gene knockouts (n=14) and controls (n=14) became hypertensive, but the former required 15.6±2.5 days versus 29.3±1.4 days for the controls ( P P 2A -AR subtype exerts a sympathoinhibitory effect, and its loss leads to a hypertensive, hyperadrenergic state.

Role of α2-Adrenergic Receptor Subtypes in the Acute Hypertensive Response to Hypertonic Saline Infusion in Anephric Mice

Experimental evidence suggests that the acute hypertensive response induced in anephric animals by infusion of a hypertonic saline solution is mediated by disinhibition of the presynaptic sympathoinhibitory alpha(2)-adrenergic receptors (alpha(2)-AR) of the central nervous system. The purpose of the present experiments was to dissect the role of the 3 distinct alpha(2)-AR subtypes (alpha(2A)-, alpha(2B), - and alpha(2C)-AR) in this response. Groups of genetically engineered mice deficient in each one of these alpha(2)-AR subtype genes were submitted to bilateral nephrectomy followed by a 0.4-mL infusion of 4% saline over a 2-hour period, with constant direct blood pressure (BP) monitoring. The alpha(2A)-AR-deficient and alpha(2C)-AR-deficient mice responded with significant BP elevations (by 11.8+/-2.5 and 16.7+/-1.7 mm Hg, respectively), and so did their wild-type counterparts (17.8+/-2.5 and 11.8+/-2.0 mm Hg, respectively) and the wild-type alpha(2B) +/+ (13.1+/-2.4 mm Hg). However, the alpha(2B)-AR-deficient mice were unable to raise their BP and had a slightly lowered BP (by -3.0+/-4. 0 mm Hg) at the end of the infusion period. All 6 groups exhibited elevated plasma norepinephrine levels ranging between 0.8 and 1.8 ng/mL at the end of the infusion. In all cases, the alpha(2)-AR-deficient groups tended to have higher norepinephrine levels than their wild-type counterparts. Surprisingly, this difference was significant only in the alpha(2B)-AR-deficient mice, which, despite the elevated norepinephrine, were unable to raise their BP. The data suggest that a full complement of the alpha(2B)-AR is needed to mediate the hypertensive response to acute saline load, even though its absence does not prevent the release of norepinephrine under these conditions.

Role of the postsynaptic α2-adrenergic receptor subtypes in catecholamine-induced vasoconstriction

Catecholamines induce direct vasoconstriction mediated by postsynaptic alpha-adrenergic receptors (alpha-ARs) of both the alpha(1) and alpha(2) type. To evaluate the contribution of each alpha(2)-AR subtype (alpha(2A), alpha(2B), and alpha(2C)) to this function, we used groups of genetically engineered mice deficient for the gene to each one of these subtypes and compared their blood pressure (BP) responses to their wild-type counterparts. Blood pressure responses to a bolus of norepinephrine (NE) were assessed before and after sequential blockade of alpha(1)-ARs with prazosin and alpha(2)-ARs with yohimbine. The first NE bolus elicited a brief 32 to 44 mm Hg BP rise (p < 0.001 from baseline) in all six groups. Prazosin decreased BP by 23 to 33 mm Hg in all groups, establishing a new lower baseline. Repeat NE at that point elicited lesser but still significant (p < 0.001) brief pressor responses between 32% and 45% of the previous BP rise in five of the six groups. Only the alpha(2A)-AR gene knockouts differed, responding instead with a 20-mm Hg fall in BP, a significant change from baseline (p < 0.001) and different from the pressor response of their wild-type counterparts (p < 0.001). The addition of yohimbine produced no further BP change in the five groups, but it did produce a small 7. 5-mm Hg fall (p < 0.05) in the alpha(2A)-AR knockouts. Norepinephrine bolus during concurrent alpha(1) and alpha(2)-AR blockade produced significant (p < 0.001) hypotensive responses in all subgroups, presumably attributable to unopposed stimulation of beta(2)-vascular wall ARs. We conclude that the alpha(2)-AR-mediated vasoconstriction induced by catecholamines is attributable to the alpha(2A)-AR subtype because mice deficient in any one of the other subtypes retained the capacity for normal vasoconstrictive responses. However, the alpha(1)-ARs account for the major part (as much as 68%) of catecholamine-induced vasoconstriction.

Mechanisms Mediating the Vasoactive Effects of the B1 Receptors of Bradykinin

Abstract—Bradykinin normally exerts its vasodilatory effect via the B2 receptor (B2R), but in this receptor’s absence, the B1 receptor becomes expressed and activated. To explore the mechanism of B1R-mediated vasodilation, 8 groups of B2R gene–knockout mice received a 2-week infusion of a B1R antagonist (300 &mgr;g · kg−1 · d−1) or vehicle (groups 1 and 2), B1R antagonist or vehicle plus NO inhibition with N&ohgr;-nitro-l-arginine methyl ester (groups 3 and 4), B1R antagonist or vehicle plus cyclooxygenase inhibition with indomethacin (groups 5 and 6), or B1R antagonist or vehicle plus blockade of vasoconstricting prostaglandin (PG) H2 and thromboxane A2 (TxA2) with SQ29548 (groups 7 and 8). The B1R antagonist produced significant (P <0.05) blood pressure increases of 17.7±3.1 mm Hg in group 1 and 10.4±3 mm Hg in group 3, whereas their vehicle-treated respective control groups 2 and 4 had no significant blood pressure changes. Indomethacin abolished the capacity of the B1R antagonist to raise blood pressure, as did blockade of the receptors of PGH2 and TxA2. Injection with the B1R agonist produced a hypotensive response (12±1.3 mm Hg), which was further accentuated by TxA2 blockade (21.7±4.1 mm Hg). Analysis of B1R gene expression by reverse transcription–polymerase chain reaction (PCR) in cardiac and renal tissues revealed marked expression at baseline, with further upregulation by 1.5- to 2-fold after various manipulations. Expression of the TxA2 receptor gene in renal tissue by quantitative real-time PCR was significantly lower in mice treated with the B1R antagonist, consistent with increased levels of agonist for this receptor. The data confirm that the B1R becomes markedly expressed in the absence of B2R and suggest that it contributes to vasodilation by inhibiting a vasoconstricting product of the arachidonic acid cascade acting via the PGH2/TxA2 receptor.

Expression of α2-Adrenergic Receptors in Normal and Atherosclerotic Rabbit Aorta

Alpha2-adrenergic receptors (alpha2-ARs) in vascular smooth muscle cells are known to mediate vasoconstriction; however, it is unknown which of the 3 subtypes of alpha2-AR (alpha2A, alpha2B, or alpha2C) is expressed in vascular tissue. We have used subtype-specific probes in in situ hybridization and RNase protection assays to analyze the expression of alpha2-AR in the thoracic aorta of New Zealand White (NZW) and Watanabe heritable hyperlipidemic (WHHL) rabbits, a model for atherosclerosis. We found that the alpha2A-AR mRNA was in endothelial and smooth muscle cells in both NZW and WHHL aorta. In addition, the shoulders and subendothelial regions of the atherosclerotic lesions in WHHL aorta showed abundant expression of alpha2A-AR mRNA. Antibodies to macrophage (RAM-11) and smooth muscle cell (HHF-35) antigens were used to localize macrophage and smooth muscle cells in aortic sections from WHHL rabbits. The expression of alpha2A-AR mRNA within the lesions of WHHL rabbits correlated with the presence of infiltrating macrophages. We discuss the potential role of alpha2A-ARs in macrophage function and in promoting atherosclerosis.