Physiology and pharmacology of the vanilloid receptor

The identification and cloning of the vanilloid receptor 1 (TRPV1) represented a significant step for the understanding of the molecular mechanisms underlying the transduction of noxious chemical and thermal stimuli by peripheral nociceptors. TRPV1 is a non-selective cation channel gated by noxious heat, vanilloids and extracellular protons. TRPV1 channel activity is remarkably potentiated by pro-inflammatory agents, a phenomenon that is thought to underlie the peripheral sensitisation of nociceptors that leads to thermal hyperalgesia. Cumulative evidence is building a strong case for the involvement of this receptor in the etiology of both peripheral and visceral inflammatory pain, such as inflammatory bowel disease, bladder inflammation and cancer pain. The validation of TRPV1 receptor as a key therapeutic target for pain management has thrust intensive drug discovery programs aimed at developing orally active antagonists of the receptor protein. Nonetheless, the real challenge of these drug discovery platforms is to develop antagonists that preserve the physiological activity of TRPV1 receptors while correcting over-active channels. This is a condition to ensure normal pro-prioceptive and nociceptive responses that represent a safety mechanism to prevent tissue injury. Recent and exciting advances in the function, dysfunction and modulation of this receptor will be the focus of this review.     

Molecular pharmacology of the human prostaglandin D2 receptor,CRTH2

1. The recombinant human prostaglandin D(2) (PGD(2)) receptor, hCRTH2, has been expressed in HEK293(EBNA) and characterized with respect to radioligand binding and signal transduction properties. High and low affinity binding sites for PGD(2) were identified in the CRTH2 receptor population by saturation analysis with respective equilibrium dissociation constants (K(D)) of 2.5 and 109 nM. This revealed that the affinity of PGD(2) for CRTH2 is eight times less than its affinity for the DP receptor. 2. Equilibrium competition binding assays revealed that of the compounds tested, only PGD(2) and several related metabolites bound with high affinity to CRTH2 (K(i) values ranging from 2.4 to 34.0 nM) with the following rank order of potency: PGD(2)>13,14-dihydro-15-keto PGD(2)>15-deoxy-Delta(12,14)-PGJ(2)>PGJ(2)>Delta(12)-PGJ(2)>15(S)-15 methyl-PGD(2). This is in sharp contrast with the rank order of potency obtained at DP : PGD(2)>PGJ(2)>Delta(12)-PGJ(2)>15-deoxy-Delta(12,14)-PGJ(2) >13,14-dihydro-15-keto-PGD(2). 3. Functional studies demonstrated that PGD(2) activation of recombinant CRTH2 results in decrease of intracellular cAMP in a pertussis toxin-sensitive manner. Therefore, we showed that CRTH2 can functionally couple to the G-protein G(alphai/o). PGD(2) and related metabolites were tested and their rank order of potency followed the results of the membrane binding assay. 4. By Northern blot analysis, we showed that, besides haemopoietic cells, CRTH2 is expressed in many other tissues such as brain, heart, thymus, spleen and various tissues of the digestive system. In addition, in situ hybridization studies revealed that CRTH2 mRNA is expressed in human eosinophils. Finally, radioligand binding studies demonstrated that two eosinophilic cell lines, butyric acid-differentiated HL-60 and AML 14.3D10, also endogenously express CRTH2.     

Prostaglandin J2 family and the cardiovascular system

Prostaglandins (PGs) of the J2 family including PGJ2, delta12-PGJ2, and 15-deoxy-delta12,14-PGJ2 (15d-PGJ2) are naturally occurring metabolites of PGD2. Among them, 15d-PGJ2 is a powerful ligand for the peroxisome proliferator-activated receptor-gamma (PPARgamma). 15d-PGJ2 and synthetic PPARgamma ligands have been reported to exert several effects on vascular cells, such as anti-proliferative, differentiation-inducing, anti-apoptotic, and anti-inflammatory effects, most of which seem to be atheroprotective, although PPARgamma-independent mechanisms may be involved. Vascular endothelial cells, intimal smooth muscle cells, and cardiomyocytes express lipocalin-type PGD synthase (L-PGDS) in vivo, which catalyzes the isomeric conversion of PGH2 to PGD2. L-PGDS expression in endothelial cells is stimulated by laminar fluid shear stress. PGD2 and 15d-PGJ2 are detected in the culture medium of endothelial cells exposed to shear stress. Serum and urinary levels of L-PGDS increase in diseases with vascular injuries, such as hypertension and diabetes. Based on these findings, we hypothesize that PGs of the J2 series are physiological substances produced in the vascular wall to protect vascular cells from injurious stimuli and to repress inflammatory reactions. If this hypothesis is correct, PGJ2 family members or other similar substances may provide novel preventive and therapeutic strategies for the treatment of vascular diseases.     

Physiology and pharmacology of the (pro)renin receptor

The (pro)renin receptor [(P)RR] is a single trans-membrane domain receptor that mediates renin and prorenin specific effects. The receptor acts as co-factor for renin and prorenin by increasing their enzymatic activity on the cell-surface and it activates the mitogen activated protein kinases ERK1/2 cascade leading to cell proliferation and to up-regulation of profibrotic genes expression. Studies in genetically modified animals over-expressing (P)RR suggest a direct role for (P)RR cardiovascular and renal pathologies since rats over-expressing (P)RR in vascular smooth-muscle cells develop high blood pressure and those with an ubiquitous over-expression of (P)RR have glomerulosclerosis and proteinuria. A peptide called "handle region peptide" (HRP) mimicking part of the prosegment of prorenin was claimed to block prorenin binding to (P)RR and its activation. The mechanism of action of HRP and its specificity for (P)RR remains very controversial although infusion of this peptide gave spectacular results by preventing diabetic nephropathy in angiotensin II type1a receptor-deficient mice. In contrast to the other components of the renin angiotensin system, (P)RR is necessary to cell survival and proliferation and a mutation of (P)RR is associated with mental retardation and epilepsy, pointing to an essential role of (P)RR in brain development. The (pro)renin receptor is a more complex protein than anticipated and in depth studies of its functions that are likely not restricted to the renin angiotensin system are needed especially in the perspective of the design of a (P)RR blocker.     

Physiology and pharmacology of melatonin in relation to biological rhythms

Melatonin is an evolutionarily conserved molecule that serves a time-keeping function in various species. In vertebrates, melatonin is produced predominantly by the pineal gland with a marked circadian rhythm that is governed by the central circadian pacemaker (biological clock) in the suprachiasmatic nuclei of the hypothalamus. High levels of melatonin are normally found at night, and low levels are seen during daylight hours. As a consequence, melatonin has been called the “darkness hormone”. This review surveys the current state of knowledge regarding the regulation of melatonin synthesis, receptor expression, and function. In particular, it addresses the physiological, pathological, and therapeutic aspects of melatonin in humans, with an emphasis on biological rhythms.     

Bronchopulmonary pharmacology of some prostaglandin endoperoxide analogs in the dog.

This investigation examined quantitatively the bronchopulmonary effects of two prostaglandin endoperoxide analogs (cyclic ethers) in the spontaneously breathing, anesthetized dog. Responses to these agents were measured by computer analyses and compared to those produced by prostaglandin F2alpha (PGF2alpha). In a dose range of 0.03-1.0 mug/kg i.v., both cyclic ethers produced dose-related alterations in pulmonary airway resistance dynamic lung compliance, expiratory airflow rate, tidal volume and respiratory frequency. These pulmonary changes were significantly greater than those observed for PGF2alpha (0.3-3.0 mug/kg). The onset of activity was prompt (less than 1 min) and recovery was complete within 5-10 min. Atropine pretreatment greatly reduced the airway resistance effects of all three test agents, thus suggesting some cholinergic component to their potent bronchoconstrictor activities. These cyclic ethers are stable forms of prostaglandin endoperoxides and may represent invaluable pharmacologic tools for the further study of intermediates in prostaglandin synthesis.     

Differential sensitivity to tetrodotoxin and lack of effect of prostaglandin E2 on the pharmacology and physiology of propagated action potentials

1. We have studied the effects of prostaglandin E(2) (PGE(2)) on action potential propagation in the isolated, desheathed vagus and saphenous nerves of rats using an extracellular grease gap recording method. 2. PGE(2) evoked a small depolarization of vagus nerves but had no effect on the stimulation threshold, size or latency of either the A wave (corresponding to conduction in A fibres) or the C wave (corresponding to conduction in C fibres) of the compound action potential (CAP) recorded from either vagus or saphenous nerves. 3. Lidocaine (0.01 - 10 mM) reduced all components of the CAP of both vagus and saphenous nerves. PGE(2) had no significant effect on the sensitivity of any component of the CAP to lidocaine. 4. Tetrodotoxin (TTX, 10 microM) blocked completely both the A wave and the C wave of the CAP in either vagus or saphenous nerves. 5. In saphenous nerve preparations the A wave was blocked by lower concentrations of TTX than the C wave or any component of the CAP in vagus nerve preparations which suggests that somatosensory A fibres express a different sub-type of TTX-sensitive voltage-gated sodium channel (VGSC) than somatosensory C-fibres or visceral sensory fibres. 6. Chemical activation of VGSCs with veratridine (10 or 50 microM) induced a depolarization in either nerve. The depolarization induced by 50 microM veratridine was blocked by 10 microM TTX. 7. Although TTX-insensitive VGSCs are expressed by some vagal and some somatosensory neurones they do not appear to be expressed functionally in the axons.     

TRPM4 channels in the cardiovascular system: Physiology, pathophysiology, and pharmacology

The transient receptor potential channel (TRP) family comprises at least 28 genes in the human genome. These channels are widely expressed in many different tissues, including those of the cardiovascular system. The transient receptor potential channel melastatin 4 (TRPM4) is a Ca(2+)-activated non-specific cationic channel, which is impermeable to Ca(2+). TRPM4 is expressed in many cells of the cardiovascular system, such as cardiac cells of the conduction pathway and arterial and venous smooth muscle cells. This review article summarizes the recently described roles of TRPM4 in normal physiology and in various disease states. Genetic variants in the human gene TRPM4 have been linked to several cardiac conduction disorders. TRPM4 has also been proposed to play a crucial role in secondary hemorrhage following spinal cord injuries. Spontaneously hypertensive rats with cardiac hypertrophy were shown to over-express the cardiac TRPM4 channel. Recent studies suggest that TRPM4 plays an important role in cardiovascular physiology and disease, even if most of the molecular and cellular mechanisms have yet to be elucidated. We conclude this review article with a brief overview of the compounds that have been shown to either inhibit or activate TRPM4 under experimental conditions. Based on recent findings, the TRPM4 channel can be proposed as a future target for the pharmacological treatment of cardiovascular disorders, such as hypertension and cardiac arrhythmias.     

Physiology and pharmacology of the brushtail possum gastrointestinal tract: relationship to the human gastrointestinal tract

Oral formulations are typically based on studies from eutherian animal models. This review introduces information relating to oral formulations for a marsupial species, the Australian brushtail possum (Trichosurus vulpecula) that has arisen from research into new methods for controlling this species - a major vertebrate pest in New Zealand. Morphologically, the gastrointestinal tract of the brushtail possum is similar to that of hindgut fermenting eutherian species, but there are some striking differences in function. Limited data suggests that the pharmacokinetics and bioavailability of administered drugs are similar to that in eutherian species, but there is some evidence that possums may have specific mechanisms for handling the intake of plant toxins and xenobiotics. The development of oral formulations for a free-ranging pest species presents several challenges above those encountered in the development of therapeutic formulations for humans and domestic animals. Use of a marsupial animal model may lead to new strategies for oral formulations in humans.     

The angiotensin-converting enzyme gene family: genomics and pharmacology

Modulation of the renin-angiotensin system (RAS), and particularly inhibition of angiotensin-converting enzyme (ACE), a zinc metallopeptidase, has long been a prime strategy in the treatment of hypertension. However, other angiotensin metabolites are gaining in importance as our understanding of the RAS increases. Recently, genomic approaches have identified the first human homologue of ACE, termed ACEH (or ACE2). ACEH differs in specificity and physiological roles from ACE, which opens a potential new area for discovery biology. The gene that encodes collectrin, a homologue of ACEH, is upregulated in response to renal injury. Collectrin lacks a catalytic domain, which indicates that there is more to ACE-like function than simple peptide hydrolysis.     

15-deoxy prostaglandin J2, the nonenzymatic metabolite of prostaglandin D2, induces apoptosis in keratinocytes of human hair follicles: a possible explanation for prostaglandin D2-mediated inhibition of hair growth

Recent studies have shown that prostaglandin D2 (PGD2) and its nonenzymatic metabolite, 15-deoxy-Δ^12,14-prostaglandin J2 (15-dPGJ2), inhibit in vitro growth of explanted human hair follicles and inhibit hair growth in mice through the GPR44 (DP2). However, the underlying mechanism is still unclear. In this study, we first investigated the expression of DP2 in human hair follicles and in cultured follicular cells. We found that DP2 is strongly expressed in the outer root sheath (ORS) cells and weakly expressed in the dermal papilla (DP) cells. We observed slight growth stimulation when ORS and DP cells were treated with PGD2. We also observed slight growth stimulation when DP and ORS cells were treated with low concentrations (0.5 and 1 μM) of 15-dPGJ2. However, 5 μM 15-dPGJ2 inhibited the viability and caused apoptosis of both cell types. Exposure of cultured human hair follicles to 15-dPGJ2 resulted in significant apoptosis in follicular keratinocytes. Altogether, our data provide an evidence that 15-dPGJ2 promotes apoptosis in follicular keratinocytes and provide rationale for developing remedies for the prevention and treatment of hair loss based on DP2 antagonism.     

15-deoxy prostaglandin J2 enhances allyl alcohol-induced toxicity in rat hepatocytes.

Allyl alcohol causes hepatotoxicity that is potentiated by small doses of bacterial lipopolysaccharide (LPS) through a cyclooxygenase-2 (COX-2)-dependent mechanism. The COX-2 product prostaglandin D(2) (PGD(2)) increases hepatocyte killing by allyl alcohol in vitro. In the present study the ability of the nonenzymatic product of PGD(2), 15-deoxy-Delta12,14-prostaglandin J(2) (15d-PGJ(2)), to increase the cytotoxicity of allyl alcohol was evaluated. In a concentration-dependent manner, 15d-PGJ(2) significantly augmented cell death caused by allyl alcohol in isolated rat hepatocytes. 15d-PGJ(2) also increased the cytotoxicity of acrolein, the active metabolite of allyl alcohol. An agonist for the PGD(2) receptor neither reproduced the increase in allyl alcohol-mediated cytotoxicity nor altered the response to 15d-PGJ(2). Similarly, these responses were not affected by either an agonist or an antagonist for the peroxisome proliferator-activated receptor-gamma. The enhancement by 15d-PGJ(2) of allyl alcohol-mediated cell killing was unaffected by augmentation or inhibition of cAMP. Protein synthesis was markedly decreased by 15d-PGJ(2), but inhibition of protein synthesis alone with cycloheximide did not increase allyl alcohol-mediated cell killing. Allyl alcohol at subtoxic concentrations increased translocation of nuclear factor kappa B (NF-kappaB), whereas at cytotoxic concentrations no translocation occurred. 15d-PGJ(2) inhibited translocation of NF-kappaB from the cytosol to the nucleus both in the presence and absence of allyl alcohol. Like 15d-PGJ(2), MG132, an inhibitor of NF-kappaB activation, enhanced allyl alcohol-induced hepatocyte death. Together these results indicate that 15d-PGJ(2) augments hepatocyte killing by allyl alcohol, and the mechanism may be related to the inhibition of NF-kappaB activation.     

Separation of prostaglandin A2 and prostaglandin B2 by ion-exchange liquid chromatography

2 closely related prostaglandins (PGs), A2 and B2, were separated by ion-exchange liquid chromatography. PGA2 and PGB2 are an isomeric pair of PGs which show little resolution on thin-layer chromatography; the pair can be resolved as gas-liquid chromatography, but use of this method requires protection of the C9-carbonyl group. Ion-exchange liquid chromatography, in contrast, requires no protective derivatization to achieve separation of PGA2 and PGB2 from PGE2. 2 different column types were used, and their results compared favorably. In 1 experiment, a triethylaminoethyl cellulose column resolved the 2 species; in another experiment, a strong anion-exchange pellicular support was successful. Both columns were stable and could be used to monitor the stability of PGE2 by following the appearance of PGA2 and PGB2, using the peak height method (i.e., a plot of micrograms injected vs. peak height). Triethylaminoethyl cellulose gave somewhat better resolution of PGA2 and PGB2 as compared with the pellicular strong anion-exchange column, but the advantage of this effect is offset by the tediousness of packing columns of triethylaminoethyl cellulose.