Vasoactive potential of the b(1) bradykinin receptor in normotension and hypertension.

The B(1) type receptor of bradykinin (Bk B(1)R) is believed to be physiologically inert but highly inducible by inflammatory mediators and tissue damage. To explore the potential participation of the Bk B(1)R in blood pressure (BP) regulation, we studied mice with deleted Bk B(2)R 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 B(2)R 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 B(1)R 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 B(1)R agonist produced a dose-dependent hypotensive response in the knockout mice only. Assessment of tissue expression of B(1)R and B(2)R genes by reverse transcription-polymerase chain reaction techniques revealed significantly higher B(1)R mRNA levels in the B(2)R knockout mice at all times (normotensive baseline and hypertensive end points). At the hypertensive end points, there was always an increase in B(1)R 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 B(2)R gene expression in the wild-type mice remained unaffected by experimental manipulations. These results confirm the known vasodilatory and natriuretic function of the Bk B(2)R; they also indicate that in its absence, the B(1)R can become upregulated and assume some of the hemodynamic properties of the B(2)R. Furthermore, they indicate that experimental manipulations to produce hypertension also induce upregulation of the B(1)R, but not the B(2)R, in cardiac and renal tissues.

Effects of antisense oligodeoxynucleotide targeting of the alpha(2B)-adrenergic receptor messenger RNA in the central nervous system.

The results of previous studies with genetically engineered mice have suggested that an intact central alpha(2B)-adrenergic receptor (alpha(2B)-AR) subtype mediates the development and maintenance of salt-induced hypertension. In the present study, we sought to further define the role of this receptor by injecting antisense oligodeoxynucleotides (AS-ODNs), targeting a selected sequence of the alpha(2B)-AR mRNA, into the lateral cerebral ventricle of rats that had undergone prior subtotal nephrectomy and dietary salt loading. Cell culture studies showed that these AS-ODNs could block alpha(2B)-AR protein generation. Before AS-ODN injection, blood pressure (BP) averaged 133+/-5 mm Hg during the daytime and rose to 165+/-4 mm Hg during the nighttime activity hours (P<0.001 versus baseline average of 120+/-2 mm Hg). The injection of AS-ODNs during the early afternoon prevented the BP rise and was associated with a significant fall in heart rate (from 385+/-12 to 306+/-15 bpm, P<0.05) and symptoms of sedation that lasted for several hours, with a peak at 3 to 6 hours and full recovery by 24 hours. At that time, a second injection produced identical effects in all rats (n=9). Control rats (n=10) that received scrambled ODN injections had no changes in BP or heart rate patterns, and neither group had evidence of neurotoxicity, indicating that these effects are specifically due to translational inhibition of central alpha(2B)-AR. We conclude that a fully functional central alpha(2B)-AR is necessary for the induction of salt-dependent hypertension.

Role of the B(2) receptor of bradykinin in insulin sensitivity.

The biological actions of bradykinin (BK) are attributed to its B(2) type receptor (B(2)R), whereas the B(1)R is constitutively absent, inducible by inflammation and toxins. Previous studies in B(2)R gene knockout mice showed that the B(1)R is overexpressed, is further upregulated by hypertensive maneuvers, and assumes some of the hemodynamic functions of the B(2)R. The current experiments were designed to further clarify the metabolic function of the B(2)R and to explore whether the upregulated B(1)R can also assume the metabolic function of the missing B(2)R. One group of B(2)R-/- mice (n=9) and one of B(2)R+/+ controls (n=8) were treated for 3 days with captopril (which produced a similar blood pressure-lowering response in both groups) and studied with the hyperinsulinemic euglycemic clamp. The knockout mice had fasting and steady-state blood glucose levels similar to those of the wild-type mice but a had tendency to higher fasting insulin levels (at 27.8+/-5.2 versus 18+/-2.9 mU/L, respectively). However, they had significantly higher steady-state insulin levels (749+/-127.2 versus 429.1+/-31.5 mU/L, P<0.05) and a significantly lower glucose uptake rate (31+/-2.4 versus 41+/-2.3 mg/kg per minute, P<0.05) and insulin sensitivity index (4.6+/-0.9 versus 10+/-0.7 P<0.001). Analysis of B(1)R and B(2)R gene expression by reverse transcription-polymerase chain reaction in cardiac muscle, skeletal muscle, and adipose tissues revealed significantly higher B(1)R mRNA level in the knockouts versus wild-type (P<0.05) at baseline and a further significant upregulation in mRNA by 1.8- to 3.2-fold (P<0.05) after insulin infusion. We conclude that absence of B(2)R confers a state of insulin resistance because it results in impaired insulin-dependent glucose transport; this is probably a direct B(2)R effect because, unlike the hemodynamic autacoid-mediated effects, it cannot be assumed by the upregulated B(1)R.

Effects of ANG II on bradykinin receptor gene expression in cardiomyocytes and vascular smooth muscle cells.

Bradykinin has vasodilatory and tissue-protective effects exerted via its B(2) type receptor, whereas the B(1) receptor is constitutively absent but inducible by inflammation and toxins. In previous studies, we found that B(2) receptor gene knockout mice exhibit overexpression of the B(1) receptor, which assumes a vasodilatory function and is further upgraded in renovascular hypertension. The present study was designed to explore the effects of excess angiotensin II (ANG II) on B(1) receptor and B(2) receptor gene expression in mouse cardiomyocytes and rat vascular smooth muscle cells (VSMC) in vivo (after a 3-day infusion of 30 ng/min ANG II in 11 wild-type and in 13 genetically engineered mice with deleted B(2) receptor gene) and in vitro (ANG II added in rat VSMC culture in the presence or absence of AT(1) or AT(2) receptor antagonist). Expression of B(1) and B(2) receptor mRNA was assessed by reverse transcriptase-polymerase chain reaction. ANG II infusion caused upregulation by 30% of the already significantly overexpressed B(1) receptors in cardiomyocytes of the B(2) receptor gene knockout mice, but in the wild-type mice it upregulated only the B(2) receptor mRNA by 47%. The addition of ANG II in VSMC culture produced a time-dependent induction of B(1) and upregulation of B(2) receptor gene expression, maximal at 3 h (by fivefold), declining almost to baseline by 24 h. The addition of losartan completely blocked this effect, whereas the AT(2) blocker PD-123319 made no difference, indicating that this is an AT(1)-mediated effect of ANG II. The data indicate that excess ANG II in subpressor doses in vivo upregulates expression of the B(2) receptor, but in its absence, the already overexpressed B(1) receptor is further upregulated, evidently assuming a counterregulatory response; in vitro, it transiently upregulates both bradykinin receptors.

Genetic engineering of glucose-stimulated insulin secretion in Chinese hamster ovary cells.

To engineer an a non-islet cell capable of glucose-stimulated insulin secretion, a chinese hamster ovary cell line (CHO) was transfected with a mammalian expression vector carrying the human insulin cDNA (pCB/hINS). More proinsulin than insulin was released daily by the stably transformed cell line (CHO-INS). Examination of acid-ethanol extracts confirmed that both insulin and proinsulin were stored. Immunohistochemical analysis of the cells also showed that (pro)insulin was stored. Unlike beta cells, CHO-INS cells did not secrete insulin in response to glucose. To investigate this lack of effect, we examined whether transfection of GLUT2 cDNA, which is ordinarily not expressed in CHO-INS cells, would confer glucose-stimulated insulin secretion. Consequently, we have demonstrated that glucose regulated insulin release occurs in the CHO-INS-GLUT2 cell line and that glucose potentiates the insulin secretory response to non-glucose secretagogues.