Pramod R. Saxena

Angiotensin-Converting Enzyme in the Human Heart Effect of the Deletion/Insertion Polymorphism

BACKGROUND An insertion (I)/deletion (D) polymorphism of the angiotensin-converting enzyme (ACE) gene has been associated with differences in the plasma levels of ACE as well as with myocardial infarction, cardiomyopathy, left ventricular hypertrophy, and coronary artery disease. METHODS AND RESULTS We determined the cardiac ACE activity and the ACE genotype in 71 subjects who died of noncardiac disorders. Cardiac ACE activity was significantly higher (P < .01) in subjects with the ACE DD genotype (12.7 +/- 1.9 mU/g wet wt) compared with subjects with the ID (8.7 +/- 0.8 mU/g) and the II (9.1 +/- 1.0 mU/g) genotypes. This difference was independent of sex, age, and the time required for tissue collection. CONCLUSIONS Cardiac ACE activity is highest in subjects with the DD genotype. Elevated cardiac ACE activity in these subjects may result in increased cardiac angiotensin II levels, and this may be a mechanism underlying the reported association between the ACE deletion polymorphism and the increased risk for several cardiovascular disorders.

Triptans in migraine : A comparative review of pharmacology, pharmacokinetics and efficacy

Triptans are a new class of compounds developed for the treatment of migraine attacks. The first of the class, sumatriptan, and the newer triptans (zolmitriptan, naratriptan, rizatriptan, eletriptan, almotriptan and frovatriptan) display high agonist activity at mainly the serotonin 5-HT1b and 5-HT1d receptor subtypes. As expected for a class of compounds developed for affinity at a specific receptor, there are minor pharmacodynamic differences between the triptans.Sumatriptan has a low oral bioavailability (14%) and all the newer triptans have an improved oral bioavailability and for one, risatriptan, the rate of absorption is faster. The half-lives of naratriptan, eletriptan and, in particular, frovatriptan (26 to 30h) are longer than that of sumatriptan (2h). These pharmacokinetic improvements of the newer triptans so far seem to have only resulted in minor differences in their efficacy in migraine.Double-blind, randomised clinical trials (RCTs) comparing the different triptans and triptans with other medication should ideally be the basis for judging their place in migraine therapy. In only 15 of the 83 reported RCTs were 2 triptans compared, and in 11 trials triptans were compared with other drugs. Therefore, in all placebo-controlled randomised clinical trials, the relative efficacy of the triptans was also judged by calculating the therapeutic gain (i.e. percentage response for active minus percentage response for placebo). The mean therapeutic gain with subcutaneous sumatriptan 6mg (51%) was more than that for all other dosage forms of triptans (oral sumatriptan 100mg 32%; oral sumatriptan 50mg 29%; intranasal sumatriptan 20mg 30%; rectal sumatriptan 25mg 31%; oral zolmitriptan 2.5mg 32%; oral rizatriptan 10mg 37%; oral eletriptan 40mg 37%; oral almotriptan 12.5mg 26%). Compared with oral sumatriptan 100mg (32%), the mean therapeutic gain was higher with oral eletriptan 80mg (42%) but lower with oral naratriptan 2.5mg (22%) or oral frovatriptan 2.5mg (16%). The few direct comparative randomised clinical trials with oral triptans reveal the same picture. Recurrence of headache within 24 hours after an initial successful response occurs in 30 to 40% of sumatriptan-treated patients. Apart from naratriptan, which has a tendency towards less recurrence, there appears to be no consistent difference in recurrence rates between the newer triptans and sumatriptan. Rizatriptan with its shorter time to maximum concentration (tmax) tended to produce a quicker onset of headache relief than sumatriptan and zolmitriptan.The place of triptans compared with non-triptan drugs in migraine therapy remains to be established and further RCTs are required.

Cardiac Renin and Angiotensins Uptake From Plasma Versus In Situ Synthesis

The existence of a cardiac renin-angiotensin system, independent of the circulating renin-angiotensin system, is still controversial. We compared the tissue levels of renin-angiotensin system components in the heart with the levels in blood plasma in healthy pigs and 30 hours after nephrectomy. Angiotensin I (Ang I)-generating activity of cardiac tissue was identified as renin by its inhibition with a specific active site-directed renin inhibitor. We took precautions to prevent the ex vivo generation and breakdown of cardiac angiotensins and made appropriate corrections for any losses of intact Ang I and II during extraction and assay. Tissue levels of renin (n = 11) and Ang I (n = 7) and II (n = 7) in the left and right atria were higher than in the corresponding ventricles (P < .05). Cardiac renin and Ang I levels (expressed per gram wet weight) were similar to the plasma levels, and Ang II in cardiac tissue was higher than in plasma (P < .05). The presence of these renin-angiotensin system components in cardiac tissue therefore cannot be accounted for by trapped plasma or simple diffusion from plasma into the interstitial fluid. Angiotensinogen levels (n = 11) in cardiac tissue were 10% to 25% of the levels in plasma, which is compatible with its diffusion from plasma into the interstitium. Like angiotensin-converting enzyme, renin was enriched in a purified cardiac membrane fraction prepared from left ventricular tissue, as compared with crude homogenate, and 12 +/- 3% (mean +/- SD, n = 6) of renin in crude homogenate was found in the cardiac membrane fraction and could be solubilized with 1% Triton X-100. Tissue levels of renin and Ang I and II in the atria and ventricles were directly correlated with plasma levels (P < .05), and in both tissue and plasma the levels were undetectably low after nephrectomy. We conclude that most if not all renin in cardiac tissue originates from the kidney. Results support the contentions that in the healthy heart, angiotensin production depends on plasma-derived renin and that plasma-derived angiotensinogen in the interstitial fluid is a potential source of cardiac angiotensins. Binding of renin to cardiac membranes may be part of a mechanism by which renin is taken up from plasma.

Coronary Side-Effect Potential of Current and Prospective Antimigraine Drugs

BACKGROUND The antimigraine drugs ergotamine and sumatriptan may cause angina-like symptoms, possibly resulting from coronary artery constriction. We compared the coronary vasoconstrictor potential of a number of current and prospective antimigraine drugs (ergotamine, dihydroergotamine, methysergide and its metabolite methylergometrine, sumatriptan, naratriptan, zolmitriptan, rizatriptan, avitriptan). METHODS AND RESULTS Concentration-response curves to the antimigraine drugs were constructed in human isolated coronary artery segments to obtain the maximum contractile response (Emax) and the concentration eliciting 50% of Emax (EC50). The EC50 values were related to maximum plasma concentrations (Cmax) reported in patients, obtaining Cmax/EC50 ratios as an index of coronary vasoconstriction occurring in the clinical setting. Furthermore, we studied the duration of contractile responses after washout of the acutely acting antimigraine drugs to assess their disappearance from the receptor biophase. Compared with sumatriptan, all drugs were more potent (lower EC50 values) in contracting the coronary artery but had similar efficacies (Emax <25% of K+-induced contraction). The Cmax of avitriptan was 7- to 11-fold higher than its EC50 value, whereas those of the other drugs were <40% of their respective EC50 values. The contractile responses to ergotamine and dihydroergotamine persisted even after repeated washings, but those to the other drugs declined rapidly after washing. CONCLUSIONS All current and prospective antimigraine drugs contract the human coronary artery in vitro, but in view of low efficacy, these drugs are unlikely to cause myocardial ischemia at therapeutic plasma concentrations in healthy subjects. In patients with coronary artery disease, however, these drugs must remain contraindicated. The sustained contraction by ergotamine and dihydroergotamine seems to be an important disadvantage compared with sumatriptan-like drugs.

On serotonin and migraine: a clinical and pharmacological review

Migraine patients have chronically low systemic 5-HT, predisposing them to develop migrainous headache once an attack has been initiated. Changes in platelet 5-HT content are not causally related, but reflect similar changes at a neuronal level. Stimulation of vascular 5-HT1 receptors, probably located in the vessel wall within the dural vascular bed, may alleviate the headache and associated symptoms, but does not interact with earlier mechanisms within the pathophysiological cascade. These receptors are of an as yet unidentified 5-HT1 subtype, closely resembling, but not identical to 5-HT1D receptors. Activation of these receptors results in vasoconstriction, inhibiting depolarization of sensory perivascular afferents within the trigemino-vascular system and thus stopping the headache. Additional inhibition of the release of vasoactive neuropeptides may be involved, but seems to be of only secondary clinical importance.

Angiotensin II Type 2 Receptor–Mediated Vasodilation in Human Coronary Microarteries

Background—Angiotensin (Ang) II type 2 (AT2) receptor stimulation results in coronary vasodilation in the rat heart. In contrast, AT2 receptor–mediated vasodilation could not be observed in large human coronary arteries. We studied Ang II–induced vasodilation of human coronary microarteries (HCMAs). Methods and Results—HCMAs (diameter, 160 to 500 μm) were obtained from 49 heart valve donors (age, 3 to 65 years). Ang II constricted HCMAs, mounted in Mulvany myographs, in a concentration-dependent manner (pEC50, 8.6±0.2; maximal effect [Emax], 79±13% of the contraction to 100 mmol/L K+). The Ang II type 1 receptor antagonist irbesartan prevented this vasoconstriction, whereas the AT2 receptor antagonist PD123319 increased Emax to 97±14% (P <0.05). The increase in Emax was larger in older donors (correlation ΔEmax versus age, r =0.47, P <0.05). The PD123319-induced potentiation was not observed in the presence of the NO synthase inhibitor L-NAME, the bradykinin type 2 (B2) receptor antagonist Hoe140, or after removal of the endothelium. Ang II relaxed U46619-preconstricted HCMAs in the presence of irbesartan by maximally 49±16%, and PD123319 prevented this relaxation. Finally, radioligand binding studies and reverse transcription–polymerase chain reaction confirmed the expression of AT2 receptors in HCMAs. Conclusions—AT2 receptor–mediated vasodilation in the human heart appears to be limited to coronary microarteries and is mediated by B2 receptors and NO. Most likely, AT2 receptors are located on endothelial cells, and their contribution increases with age.

The current endothelin receptor classification: time for reconsideration?

The possible involvement of endothelins in a variety of diseases has attracted the attention of many pharmacologists in search of a novel therapeutic approach. The rapid development of endothelin research has resulted in the molecular characterization and pharmacological recognition of ETA and ETB receptors, and in the development of compounds selective for these receptors. However, the characterization of receptors in various assays has shown that a number of effects are mediated by receptors that do not fit the present criteria for ETA or ETB receptors. In this article, Willem Bax and Pramod Saxena address endothelin receptors in general, and atypical receptors in particular.

Migraine headache is not associated with cerebral or meningeal vasodilatation—a 3T magnetic resonance angiography study

Sir, with great interest, we read the article by Schoonman et al. (2008), who claim that ‘in contrast to widespread belief, migraine attacks are not associated with vasodilatation of cerebral or meningeal blood vessels’ and, therefore, ‘future anti-migraine agents may not require vasoconstrictor action’. Whereas the methodology used in this investigation is elegant, we disagree with their claim for the following reasons. First, it may be noted that Schoonman et al. (2008) did observe vasodilatation during nitroglycerin (NTG; 0.5 mg/kg/min over 20 min) infusion in all blood vessels investigated (middle meningeal, internal and external carotid, middle cerebral, basilar and posterior cerebral arteries), but not during the migraine-like headache provoked 1.5–5.5 h after cessation of NTG infusion. However, these initial vasodilator changes can trigger a chain of events leading to headache. Cranial vasodilatation leads to enhanced blood volume following each cardiac stroke with consequent augmentation of vascular pulsations, which may be sensed by stretch receptors in the vessel wall; the resultant increase in perivascular (trigeminal) sensory nerve activity can provoke headache and other migraine symptoms (Fig. 2; Villalón et al., 2003). Admittedly, the magnitude of vasodilatation did not significantly differ between the 20 subjects later developing a migraine-like attack after NTG and the 7 subjects who did not or between the two sides in case of unilateral headache. This may, however, be due to differences in headache threshold. Second, Schoonman et al. (2008) measured diameter changes in the proximal (extracranial) conducting section of the meningeal artery, while the distal intracranial regions could not be studied due to technical reasons. The authors submit that ‘it seems likely that there were also no changes in the intracranial resistance microvasculature during migraine attacks, [because] basilar and internal carotid artery blood flows, . . . which are dependent on cardiac output, arterial calibre and vasomotor tone in small resistance vessels, . . . did also not change during migraine attacks’. It should be obvious that basilar and internal carotid artery blood flows do not directly relate to diameter changes in the middle meningeal artery, which originates from the external carotid artery. Moreover, there may be regional differences in the innervation of the meningeal artery (O’Connor and van der Kooy, 1986; Strassman et al., 2004), which illustrates that it is an oversimplification to consider the large extracranial section of the meningeal artery as an accurate representative of its smaller, intracranial portion. Indeed, as described for the coronary circulation in detail (for a review, see Tiefenbacher and Chilian, 1998), pharmacological responses to vasoactive compounds may differ between proximal and distal regions of the meningeal artery. For example, we demonstrated that large ‘conducting’ portions of the porcine-isolated meningeal artery are insensitive to the 5-HT1B/1D receptor agonist sumatriptan (Mehrotra et al., 2006), while others observed contractions when the whole meningeal arterial bed, including resistance vessels, was perfused (Bou et al., 2000). In addition, there is at least some evidence for the dilatation of cranial extracerebral arteriovenous anastomoses in migraine (Heyck, 1969) and these shunt vessels, which were not studied here, selectively and strongly constrict in response to ergot alkaloids as well as triptans in experimental animals (Saxena and Tfelt-Hansen, 2006). Finally, one must keep in mind that this investigation concerns NTG-provoked (not spontaneous) migraine headaches and lacks measurements at the beginning of such headaches. To their credit, Schoonman et al. (2008) have acknowledged these potential limitations. doi:10.1093/brain/awn259 Brain 2009: 132; 1–2 | e112

Prorenin, Renin, Angiotensinogen, and Angiotensin-Converting Enzyme in Normal and Failing Human Hearts Evidence for Renin Binding

BACKGROUND A local renin-angiotensin system in the heart is often invoked to explain the beneficial effects of ACE inhibitors in heart failure. The heart, however, produces little or no renin under normal conditions. METHODS AND RESULTS We compared the cardiac tissue levels of renin-angiotensin system components in 10 potential heart donors who died of noncardiac disorders and 10 subjects with dilated cardiomyopathy (DCM) who underwent cardiac transplantation. Cardiac levels of renin and prorenin in DCM patients were higher than in the donors. The cardiac and plasma levels of renin in DCM were positively correlated, and extrapolation of the regression line to normal plasma levels yielded a tissue level close to that measured in the donor hearts. The cardiac tissue-to-plasma concentration (T/P) ratios for renin and prorenin were threefold the ratio for albumin, which indicates that the tissue levels were too high to be accounted for by admixture with blood and diffusion into the interstitial fluid. Cell membranes from porcine cardiac tissue bound porcine renin with high affinity. The T/P ratio for ACE, which is membrane bound, was fivefold the ratio for albumin. Cardiac angiotensinogen was lower in DCM patients than in the donors, and its T/P ratio was half that for albumin, which is compatible with substrate consumption by cardiac renin. CONCLUSIONS These data in patients with heart failure support the concept of local angiotensin production in the heart by renin that is taken up from the circulation. Membrane binding may be part of the uptake process.

Mannose 6-Phosphate Receptor–Mediated Internalization and Activation of Prorenin by Cardiac Cells

The binding and internalization of recombinant human renin and prorenin (2500 microU/mL) and the activation of prorenin were studied in neonatal rat cardiac myocytes and fibroblasts cultured in a chemically defined medium. Surface-bound and internalized enzymes were distinguished by the addition of mannose 6-phosphate to the medium, by incubating the cells both at 37 degrees C and 4 degrees C, and by the acid-wash method. Mannose 6-phosphate inhibited the binding of renin and prorenin to the myocyte cell surface in a dose-dependent manner. At 37 degrees C, after incubation at 4 degrees C for 2 hours, 60% to 70% of cell surface-bound renin or prorenin was internalized within 5 minutes. Intracellular prorenin was activated, but extracellular prorenin was not. The half-time of activation at 37 degrees C was 25 minutes. Ammonium chloride and monensin, which interfere with the normal trafficking and recycling of internalized receptors and ligands, inhibited the activation of prorenin. Results obtained with cardiac fibroblasts were comparable to those in the myocytes. This study is the first to show experimental evidence for the internalization and activation of prorenin in extrarenal cells by a mannose 6-phosphate receptor-dependent process. Our findings may have physiological significance in light of recent experimental data indicating that angiotensin I and II are produced at cardiac and other extrarenal tissue sites by the action of renal renin and that intracellular angiotensin II can elicit important physiological responses.