Therapeutic Differences in 24-h Ambulatory Blood Pressures in Patients Switched Between Bioequivalent Nifedipine Osmotic Systems With Differing Delivery Technologies.

Comparing modified-release formulations can be difficult using current bioequivalence criteria. Two 60-mg-once-daily nifedipine formulations are deemed bioequivalent in Canada. This study examined the validity of the assumption that these interchangeable, but different, delivery technologies are therapeutically equivalent in maintaining systolic blood pressure (SBP) control throughout the entire dosing interval. We used 24-h Ambulatory Blood Pressure Monitoring to objectively examine whether formulation switches changed population SBP >2 mmHg (reflecting 6% increased stroke mortality) and in what proportion of patients SBP changed ≥6 mmHg (risking unnecessary therapeutic alterations). When 20 patients, previously receiving 60-mg-once-daily Nifedipine-GITS, were switched to Mylan-Nifedipine-XL, population-mean ± SE 24-h SBP increased 3 ± 1.1 mmHg (P = 0.0173) and 8-h nocturnal SBP increased 4 ± 1.6 mmHg (P = 0.0098). Thus, interchange of nifedipine formulations can affect therapeutic consistency. These data support existing calls to improve criteria for establishing bioequivalence between formulations employing differing modified-release technologies.

GPER-independent effects of estrogen in rat aortic vascular endothelial cells.

GPER (aka GPR30) has been identified as an important mechanism by which estrogen mediates its effects. Previous studies from our laboratories and those of others have demonstrated that GPER activation mediates a range of vascular contractile and growth regulatory responses. However, the importance of GPER in mediating the actions of estradiol (E2) in rat aortic endothelial cells is unclear. Therefore, we sought to determine the importance of GPER vs. the "classical" estrogen receptor (ER) in mediating the endothelial growth regulatory effects of E2. To do this we assessed the effect of E2 in regulating phosphoERK content and apoptotic rates in rat aortic endothelial cells and the role of GPER in mediating these effects. E2 mediated a concentration-dependent inhibition of both ERK phosphorylation and serum deprivation-induced apoptosis with a maximal effect at a concentration of 10 nM. Pretreatment with the ER antagonist ICI 182780 abolished E2-mediated inhibition of both ERK phosphorylation and apoptosis. In contrast, pretreatment with GPER antagonist G15 had no significant effect on E2-mediated inhibition of ERK phosphorylation or on apoptosis. Further, downregulation of GPER expression with a GPER shRNA adenovirus did not block E2-mediated inhibitory effects on ERK phosphorylation and apoptosis. In fact, these inhibitory effects of E2 were further enhanced by GPER downregulation. Downregulation of ERα expression reversed the E2-mediated inhibitory effects to stimulatory effects. E2's phosphoERK and apoptosis stimulatory effects seen with ERα downregulation are attenuated by pretreatment with G15. In conclusion, in rat aortic endothelial cells, E2-mediated endothelial effects are predominantly driven by ER and not by GPER.

Intraclass differences among antihypertensive drugs.

The four major classes of antihypertensive drugs­diuretics, ß-blockers, calcium channel blockers, and renin-angiotensin system inhibitors (including angiotensin-converting enzyme inhibitors and angiotensin receptor blockers)­have significant qualitative and quantitative differences in the adverse effects they cause. Structural and chemical differences have been identified within these classes, especially among the calcium channel blockers and, to a lesser extent, among the thiazide/thiazide-like diuretics. However, it has been more difficult to demonstrate that these differences translate into differential effects with respect to either the surrogate endpoint of blood pressure reduction or, more importantly, hypertension-related cardiovascular complications. Based on a hierarchy-of-evidence approach, differences are apparent between hydrochlorothiazide and chlorthalidone based on evidence of moderate quality. Low-quality evidence suggests atenolol is less effective than other ß-blockers. However, no significant intraclass differences have been established among the other classes of antihypertensive drugs.

Hypertension management in patients with renal cell cancer treated with anti-angiogenic agents.

Inhibitors of the vascular endothelial growth factor (vegf-is) signalling pathway have fundamentally changed the treatment of metastatic renal cell carcinoma (mrcc). Hypertension is one of the most common side effects of vegf-is and has been reported with almost every vegf-i used for treatment to date. The exact mechanism of vegf-i-induced hypertension appears complex and multifactorial, and it remains to be fully explained. No randomized clinical trials are available to guide the management of hypertension during vegf-i treatment in mrcc patients. The guiding principles suggested here summarize the consensus of opinions on the diagnosis and management of vegf-i-induced hypertension during treatment of mrcc obtained from an expert working group composed of 4 Canadian medical oncologists and 5 Canadian hypertension specialists. The Canadian Hypertension Education Program guidelines, available literature, and expert opinion were used to develop the guiding principles.

Impact of age and gender on blood pressure and low-density lipoprotein cholesterol reduction: results of a pooled analysis.

OBJECTIVES: To compare the simultaneous reduction of blood pressure (BP) to below 150 mmHg and low-density lipoprotein cholesterol (LDL-C) after treatment with single-pill amlodipine/atorvastatin (SPAA) among younger (<65 years), older (≥65 years) and elderly (≥75 years) men and women with hypertension and dyslipidemia. METHODS: Data from five, 14-20-week, open-label, multi-national studies (GEMINI US, GEMINI-Australia, Asia, Latin-America, Africa/Middle-East [AALA], JEWEL 1, JEWEL 2, and the Clinical Utility of Caduet in Simultaneously Achieving Blood Pressure and Lipid End Points [CAPABLE]) were pooled. In these studies, SPAA (5/10 to 10/80 mg/mg) was electively titrated to achieve study-specific targets. Reductions in BP and LDL-C, and changes in renal and liver function tests, fasting glucose and adverse event (AE) rates were compared across the three age groups. RESULTS: A total of 3613 patients (65%) were <65 years, 1946 (35%) were ≥65 years and 441 (8%) were ≥75 years. Baseline mean systolic BP tended to increase with age and diastolic BP and LDL-C decreased, p<0.001. Final mean SPAA dose was similar (7.2/23.9, 7.1/24.3, 7.1/24.0 mg/mg). Final mean BP in the younger/older/elderly groups was 128.1/79.9, 131.3/75.0, 132.8/73.4 mmHg (adjusted BP reductions -20.2/-10.4, -18.6/-12.7, -17.7/-13.2 mmHg, p<0.001). Final mean LDL-C was 91, 87, 87 mg/dl (2.4, 2.3, 2.3 mmol/l) p<0.001; adjusted %LDL-C reductions -27.1, -26.8, -26.4, p<0.001. Estimated glomerular filtration rate increased in the younger group but decreased in the older and elderly groups (p=0.005). Small increases in liver function tests and fasting glucose were observed. Discontinuations due to AEs tended to increase with age but were low in all groups (6.2%, 7.9%, 8.8%, p=0.045). Study limitations include post hoc analysis and short duration of follow-up. CONCLUSIONS: Simultaneous reduction of BP to below 150 mmHg and LDL-C using SPAA is both effective and well-tolerated among younger and older men and women, including those aged≥75 years. Clinicians may be reassured by the low proportion of AEs that led to discontinuation in all groups suggesting that older patients were not disadvantaged by this treatment.

Analysis of a missense variant of the human N-formyl peptide receptor that is associated with agonist-independent beta-arrestin association and indices of inflammation.

Formyl-Met-Leu-Phe (fMLP) is a potent chemoattractant molecule released from both bacteria and damaged mitochondria that activates fMLP receptors (FPR) leading to neutrophil chemotaxis, degranulation and superoxide production. A common missense single nucleotide polymorphism in the human FPR1 gene at nucleotide c.32C>T results in the amino-acid substitution, p.I11T, in the FPR1 extracellular amino-terminus. The minor (c.32T) allele frequencies were 0.25, 0.27, 0.25, 0.15 and 0.14 in healthy Caucasian, African, East Indian, Chinese and Native Canadian individuals, respectively. In subjects homozygous for the p.T11 allele, we find elevated serum concentrations of C-reactive protein, increased absolute counts of blood leukocytes and neutrophils, and erythrocyte sedimentation rates. When expressed in HEK 293 and RBL-2H3 cells a substantial proportion of FPR1 p.I11T variant is retained intracellularly and agonist-independent internalization of the FPR1 p.I11T variant, but not the wild-type FPR1, is constitutively associated with beta-arrestin2-GFP in vesicles. Moreover, basal N-acetyl-D-glucosaminidase release is increased in primary neutrophils isolated from subjects either heterozygous or homozygous for the FPR1 p.T11 allele. Taken together, the data suggest an increased receptor activity and phenotypic expression of increased inflammatory indices in subjects with the p.T11 allele.

Tyrosine kinase-mediated serine phosphorylation of adenylyl cyclase.

Receptor tyrosine kinase (RTK) activation is associated with modulation of heptahelical receptor-stimulated adenylyl cyclase responses. The mechanisms underlying the RTK-mediated enhancement of adenylyl cyclase function remain unclear. In the present studies, we show that the tyrosine kinase-dependent enhancement of adenylyl cyclase isoform VI function parallels an enhancement in serine phosphorylation of the enzyme. This effect was mediated by both RTK activation, with IGF-1, and by tyrosine phosphatase inhibition, with sodium orthovanadate. This enhancement of adenylyl cyclase function was not attenuated by inhibitors of ERK, PKC, PKA, or PI3 kinase activity but was blunted by inhibition of endogenous p74(raf-1)() activity. To characterize the molecular site of this effect we identified multiple candidate serine residues in and adjacent to the adenylyl cyclase VI C1b catalytic region and performed serine-to-alanine site-directed mutagenesis using adenylyl cyclase VI as a template. Mutation of serine residues 603 and 608 or serine residues 744, 746, 750, and 754 attenuated both the tyrosine kinase-mediated enhancement of enzyme phosphorylation as well as the sensitization of function. Together, these data define a novel tyrosine kinase-mediated mechanism leading to serine phosphorylation of adenylyl cyclase isoform VI and the sensitization of adenylyl cyclase responsiveness.

The 2000 Canadian recommendations for the management of hypertension: Part one--therapy.

OBJECTIVE: To provide updated, evidence-based recommendations for the therapy of hypertension in adults. OPTIONS: For patients with hypertension, there are a number of lifestyle manoeuvres and antihypertensive agents that may control blood pressure. Randomized trials evaluating first- line therapy with thiazides, beta-adrenergic antagonists, angiotensin-converting enzyme inhibitors, calcium channel blockers, alpha-blockers, centrally acting agents or angiotensin II receptor antagonists were reviewed. OUTCOMES: The health outcomes considered were changes in blood pressure, cardiovascular morbidity, and cardiovascular and/or all-cause mortality rates. Economic outcomes were not considered due to insufficient evidence. EVIDENCE: Medline searches were conducted from the period of the last revision of the Canadian Recommendations for the Management of Hypertension (May 1998 to October 2000). Reference lists were scanned, experts were polled, and the personal files of the subgroup members and authors were used to identify other studies. All relevant articles were reviewed and appraised, using prespecified levels of evidence, by content experts and methodological experts. VALUES: A high value was placed on the avoidance of cardiovascular morbidity and mortality. BENEFITS, HARMS, AND COSTS: Various lifestyle manoeuvres and antihypertensive agents reduce the blood pressure of patients with sustained hypertension. In certain settings, and for specific classes of drugs, blood pressure lowering has been associated with reduced cardiovascular morbidity and/or mortality. RECOMMENDATIONS: The present document contains detailed recommendations pertaining to all aspects of the therapy of patients with hypertension, including lifestyle modifications proven to lower blood pressure, treatment thresholds, target blood pressures, choice of agents in various settings and strategies to enhance adherence. Lower thresholds for blood pressure treatment are advocated for people with other cardiovascular risk factors or established hypertensive target organ damage. Implicit in the recommendations for therapy is the principle that treatment should be individualized for each patient and the choice of agent should be dictated by coexistent conditions. For the treatment of uncomplicated essential hypertension, thiazides, beta-adrenergic antagonists, angiotensin-converting enzyme inhibitors or calcium channel blockers may be appropriate, depending on individual circumstances. VALIDATION: All recommendations were graded according to strength of the evidence and voted on by the Canadian Hypertension Recommendations Working Group. Only those recommendations achieving high levels of consensus are reported here. These guidelines will be updated annually.

Quinapril treatment enhances vascular sensitivity to insulin.

OBJECTIVE: Insulin-mediated vasodilation has been shown to be impaired with hypertension and aggravated by dietary sodium restriction. Whether vascular insulin resistance in this setting could be improved by antihypertensive therapy was unknown. Therefore, we determined the effect of therapy with an angiotensin-converting enzyme inhibitor, on vascular sensitivity to insulin in hypertensive subjects fed a sodium-restricted diet. DESIGN AND METHODS: The effects of 3 months of therapy with quinapril on vascular sensitivity to insulin was assessed in 11 hypertensive subjects using a randomized, placebo-controlled, double-blind design. Vascular sensitivity to insulin was assessed by determination of the insulin ED50 from dorsal hand vein linear variable differential transformer in phenylephrine-preconstricted vessels. Subjects were maintained on a 75 mmol/day sodium diet for 3 days prior to each study. RESULTS: Quinapril therapy significantly improved vascular sensitivity to insulin, as assessed by a decrease in the ED50 for insulin (ED50 insulin: placebo = 501 +/- 189 [muU/ min; quinapril = 276 +/- 100 muU/min P < 0.05). Isoproterenol-mediated relaxation was also enhanced by quinapril treatment (maximal isoproterenol-mediated relaxation: Placebo = 92 +/- 15% of baseline distension; Quinapril = 151 +/- 31% P < 0.05). CONCLUSIONS: The current study suggests the hypothesis that the ACE-inhibitor quinapril has beneficial effects on vascular function in general, and on insulin-mediated vascular responses, in particular.

G-Protein-coupled receptor kinase activity in hypertension : increased vascular and lymphocyte G-protein receptor kinase-2 protein expression.

Impaired receptor-stimulated adenylyl cyclase activation has been observed in lymphocytes from hypertensive subjects and has been linked to an increase in lymphocyte G-protein receptor kinase-2 (GRK-2) protein expression. However, whether the increase in lymphocyte GRK-2 reflected an increase in vascular GRK-2 was unknown. Therefore, we compared GRK-2 protein expression in lymphocytes and aortas obtained from normotensive Wistar rats, Wistar-Kyoto rats (WKY), and spontaneously hypertensive rats (SHR) and from aortas of Dahl rats. Impaired beta-adrenergic responsiveness was observed in lymphocytes and vascular tissues obtained from hypertensive SHR (10 and 15 weeks old) but not in those obtained from prehypertensive SHR (5 weeks old). Immunodetectable lymphocyte GRK-2 protein expression was increased in 10-week-old SHR (143+/-10% of the expression in 10-week-old Wistar rats and 131+/-11% of the expression in 10-week-old WKY, n=5 in each group). Immunodetectable vascular smooth muscle cell GRK-2 was comparably increased (169+/-14% of the expression in Wistar rats and 138+/-7% of the expression in WKY, n=5 in each group). Also, in hypertensive Dahl salt-sensitive rats, vascular GRK-2 protein expression was increased (185+/-14% of the expression in Dahl salt-resistant rats, n=5 in each group) compared with Dahl salt-resistant controls. These studies support a generalized defect in vascular GRK-2 protein expression in hypertension, which could be an important factor in the impairment of beta-adrenergic-mediated vasodilation, characteristic of the hypertensive state.

Regulation of tyrosine kinase activation and granule release through beta-arrestin by CXCRI.

Chemoattractant-stimulated granule release from neutrophils, basophils and eosinophils is critical for the innate immune response against infectious bacteria. Interleukin 8 (IL-8) activation of the chemokine receptor CXCRI was found to stimulate rapid formation of beta-arrestin complexes with Hck or c-Fgr. Formation of beta-arrestin-Hck complexes led to Hck activation and trafficking of the complexes to granule-rich regions. Granulocytes expressing a dominant-negative beta-arrestin-mutant did not release granules or activate tyrosine kinases after IL-8 stimulation. Thus, beta-arrestins regulate chemokine-induced granule exocytosis, indicating a broader role for beta-arrestins in the regulation of cellular functions than was previously suspected.

Identification of a novel mechanism for endotoxin-mediated down-modulation of CC chemokine receptor expression.

In the present study, we explored the molecular mechanisms by which bacterial endotoxin (LPS) mediates the down-regulation of CCR2 receptors on human monocytes. We found that LPS induced a marked reduction in CCR2 cell surface protein levels which was blocked by pretreatment with the tyrosine kinase inhibitors genistein and herbimycin A. The effector mechanism underlying LPS-induced CCR2 down-modulation appears to involve the enzymatic activity of proteinases since Western blot analysis of LPS-stimulated monocytes revealed the degradation of a 38-kDa species corresponding to the CCR2B monomer. In RBL cells expressing the CCR2B-green fluorescent protein (GFP) fusion chemokine receptor, LPS stimulated the internalization and degradation of CCR2. The serine proteinase inhibitor N-alpha-p-tosyl-L-lysine chloromethyl ketone blocked LPS-induced down-modulation of CCR2 in monocytes and CCR2B-GFP in RBL cells. This work describes a previously uncharacterized mechanism for CC chemokine receptor down-modulation that is dependent upon tyrosine kinase activation and serine proteinase-mediated receptor degradation and may provide further insight into the mechanisms of leukocyte regulation during immunological and inflammatory responses.

Vanadate stimulation of adenylyl cyclase: an index of tyrosine kinase vascular effects.

BACKGROUND: Beyond their mitogenic effects, hormones such as insulin, which activate receptor tyrosine kinases, regulate vascular tone. Further, we have demonstrated that receptor tyrosine kinase activation enhances adenylyl cyclase activation, a prominent mechanism that mediates vasodilation. However, whether tyrosine kinase-mediated human vascular responses parallel tyrosine kinase-mediated cellular effects on adenylyl cyclase activity is unknown. METHODS AND RESULTS: To assess tyrosine kinase-mediated vascular responses, vascular sensitivity to insulin was assessed with the dorsal hand vein linear variable differential transformer technique. Insulin infusion resulted in a dose-dependent relaxation in all subjects. Cellular responses were assessed by means of the insulinomimetic vanadate-mediated sensitization of vascular adenylyl cyclase activity. Vanadate stimulated a tyrosine kinase-dependent enhancement of adenylyl cyclase function in human and rat aortic vascular smooth muscle cells, human lymphocytes, and human aortic endothelial cells. Further, maximal insulin-mediated vasodilation was significantly positively correlated with maximal vanadate-mediated enhancement of human lymphocyte adenylyl cyclase activity. CONCLUSION: Insulin-mediated vasodilation is positively correlated with vanadate-mediated enhancement of adenylyl cyclase activity. Vanadate-mediated enhancement of adenylyl cyclase activity in lymphocytes may represent an index of tyrosine kinase-mediated vascular effects.

HMO consolidations: how national mergers affect local markets.

The health maintenance organization (HMO) industry has undergone a wave of national consolidations in recent years. The most notable among these were between United HealthCare and MetraHealth (1995), PacifiCare Health Systems and FHP International (1996), Aetna Life and Casualty and U.S. Healthcare (1996), and Aetna and Prudential's health care unit (1999). This paper examines HMO consolidation from 1994 to 1997, looking first at concentration at the national level and then at the consequences of national consolidations for local markets. Whereas earlier mergers may have caused only a small increase in the type of local market concentration that may increase prices, later and currently proposed mergers may be motivated by considerations of increasing local market concentration. However, the concentration-increasing effect of national mergers was offset by the concentration-decreasing effect of HMO entry and growth. The analyses suggest that antitrust policy still has a role to play in ensuring that HMO markets remain open to new entry and in evaluating the effect of national mergers on local market concentration.

CD45 modulation of CXCR1 and CXCR2 in human polymorphonuclear leukocytes.

All leukocytes express the cell surface glycoprotein CD45, which has intrinsic intracellular protein tyrosine phosphatase activity. CD45 is known to play a regulatory role in activation-induced signaling in lymphocytes; however, little is known of its role in non-lymphoid leukocytes. Therefore, we examined the potential effect of CD45 on chemokine-induced signaling in human neutrophils (polymorphonuclear cells, PMN). Treating isolated PMN for 2 h with an anti-CD45RB antibody (Bra11) down-modulated expression of the chemokine receptors CXCR1 and CXCR2 to 44 +/- 10% and 47 +/- 9% of their respective controls. The tyrosine kinase inhibitors genistein and herbimycin A significantly inhibited the Bra11-induced down-modulation of CXCR1 and CXCR2. Furthermore, Bra11-treated PMN were functionally inhibited in their capacity to exhibit IL-8-induced transient intracellular Ca2+ increases. Selected targeting of CXC receptors is indicated by the fact that N-formyl-Met-Leu-Phe (fMLP) receptor expression and function were not lost following Bra11 treatment. The effect of Bra11 on IL-8-mediated function and receptor expression was paralleled by decreased tyrosine phosphorylation of a 54- to 60-kDa protein. These findings indicate that CD45 can act to modulate PMN responses to chemokines; thus agents regulating CD45 can potentially modulate leukocyte traffic and may represent a novel therapeutic approach towards the treatment of inflammatory diseases.

Moderate dietary salt restriction increases vascular and systemic insulin resistance.

Our recent studies have indicated that severe salt restriction aggravates vascular insulin resistance in younger normotensive and hypertensive subjects. However, whether the extent of dietary salt restriction commonly advocated adversely affects vascular insulin resistance is unknown. To determine whether moderate dietary salt restriction might affect vascular and systemic sensitivity to insulin, we studied eight subjects after 1 week of a normal sodium diet (235 mEq/day) and 1 week of a moderate salt restriction (75 meq/day). Systemic insulin resistance as assessed by the fasting plasma glucose-to-insulin ratio was aggravated by dietary sodium restriction (normal sodium: 1.2 +/- 0.1 mmol/mIU; low sodium 0.6 +/- 0.1, P < .05). Salt restriction significantly reduced maximal insulin-mediated vasodilation (normal sodium: 51% +/- 5% of maximum nitroglycerin-mediated response; low sodium: 28% +/- 6%, P < .01). In contrast, no alterations in nitroglycerin-mediated vasodilation nor phenylephrine-mediated vasoconstriction were noted. These studies demonstrate that moderate salt restriction aggravates both systemic and vascular insulin resistance. This impairment of the vasodilating effect of insulin could be a factor attenuating the blood pressure lowering effect of a low sodium diet.

G-protein-coupled receptor kinase expression in hypertension.

In human hypertension we have recently identified an increase in lymphocyte G-protein receptor kinase-2 (GRK-2) protein expression, the key protein regulating the interaction between G-protein-coupled receptors and activation of adenylyl cyclase. However, it was not known whether this increase in GRK-2 protein expression was attributable to regulation at the level of translation. Furthermore, the relationship between extent of GRK-2 expression, receptor activation of adenylyl cyclase, and blood pressure was unclear. We therefore studied lymphocytes from 7 young subjects with borderline hypertension and 14 young normotensive subjects. Immunodetectable GRK-2 protein expression in lymphocytes from subjects with hypertension was increased (155%+/-7% of normotensive subjects; P < .05). In addition, GRK-2 protein expression was positively correlated with blood pressure (r = 0.53; P = .013) and inversely correlated with beta-adrenergic-mediated adenylyl cyclase activity (r = -0.54, P = .012). However, lymphocyte GRK-2 messenger ribonucleic acid (mRNA) content was not altered (110%+/-13% of that observed in normotensive control subjects). Increased GRK-2 protein expression may be an important factor in the impairment of beta-adrenergic-mediated vasodilation, characteristic of the hypertensive state.