Meeting the urgent need for rabies education in Haiti.

The highest rate of human rabies deaths reported in the Americas is in Haiti, and most of these deaths result from rabies virus infections that occur after individuals are bitten by infected dogs and do not receive rabies post-exposure prophylaxis. One barrier to rabies prevention in Haiti is a lack of knowledge about this disease among healthcare professionals and community members. During the past 4 years, The US Centers for Disease Control and Prevention has collaborated with public health officials and partners to develop, test and refine educational materials aimed at filling this need for rabies education. This report summarizes the use of feedback from knowledge, attitudes and practises surveys; key informant interviews; and focus groups to develop culturally appropriate rabies prevention materials for community members, health officials, clinicians, laboratory professionals, veterinary professionals, government officials and national and local district leaders about ways to prevent rabies. These formative research methods were critically important in ensuring that the materials would be culturally appropriate and would stand the greatest likelihood of motivating Haitians to protect themselves from rabies. Centers for Disease Control and Prevention is using lessons learned in Haiti to develop and test materials in other countries with high rates of canine rabies.

Inhibition of the type 1 inositol 1,4,5-trisphosphate-sensitive Ca2+ channel by calmodulin antagonists.

This study describes the effects of a number of calmodulin antagonists on the cerebellar type 1 inositol 1,4,5-trisphosphate (InsP3) receptor. All the antagonists tested (trifluoperazine, fluphenazine, chlorpromazine and calmidazolium) inhibited the extent of InsP3-induced Ca2+ release (IICR) with similar IC(50) values (between 60 and 85 microM). They did not affect the efficacy of InsP3 to release Ca2+, since the concentrations of InsP3 required to cause half-maximal release was little affected in the presence of these agents. In addition, these agents did not affect InsP3 binding to its receptor. Stopped-flow studies to determine the rate constants of IICR showed this process to be biphasic with a fast and slow component. All the calmodulin antagonists appeared to reduce the rate constants for Ca2+ release in a phase-specific manner, preferentially reducing the fast phase component. Chlorpromazine (75 microM) appeared to have the most potent effect on the fast phase rate constant, reducing it from 1.0 to 0.08 s(-1), while only reducing the rate constant for the slow phase about twofold (0.2-0.08 s(-1)). The fact that calmodulin itself inhibits both IICR and InsP3 binding, while these calmodulin antagonists also reduce Ca2+ release and do not affect InsP3 binding, suggests that the mechanism of action of these agents is unlikely to be due to the reversal of the modulatory action of calmodulin on this receptor.

Inositol 1,4,5-trisphosphate receptor isoforms show similar Ca2+ release kinetics.

The inositol 1,4,5-trisphosphate receptor (InsP3R) is an intracellular Ca2+ release channel which upon activation initiates many cellular functions. Multiple InsP3R subtypes are expressed in most cell types but the physiological significance of this heterogeneity is poorly understood. This study has directly compared the functional properties of the three different InsP3R isoforms by analyzing their InsP3-induced Ca2+ release (IICR) properties in cell lines which predominantly express each isoform subtype. The InsP3-dependence of the amount or extent of IICR was InsP3R isoform-specific, with the type III isoform having the lowest affinity with respect to Ca2+ release. The transient kinetics of IICR, measured using stopped-flow spectrofluorimetry, however, were similar for all three InsP3R isoforms. At maximal InsP3 concentrations (20 microM) the rate constants where between 0.8 and 1.0 s(-1) for the fast phase and 0.25-0.45 s(-1) for the slow phase. The concentration of InsP3 required to induce half-maximal rates of Ca2+ release (EC50) were also similar for the three isoforms (0.2-0.4 microM for the fast phase and 0.75-0.95 microM for the slow phase). These results indicate the InsP3R channel does not significantly differ functionally in terms of Ca2+ release rates between isoforms. The temporal and spatial features of intracellular Ca2+ signals are thus probably achieved through InsP3R isoform-specific regulation or localization rather than their intrinsic Ca2+ efflux properties.

Curcumin: a new cell-permeant inhibitor of the inositol 1,4,5-trisphosphate receptor.

Curcumin (diferuoylmethane or 1,7-bis (4-hydroxy-3-methoxyphenol)-1,6-hepatadiene-3,5-dione) is the active ingredient of the spice turmeric. Curcumin has been shown to have a number of pharmacological and therapeutic uses. This study shows that curcumin is a potent inhibitor of the inositol 1,4,5-trisphosphate-sensitive Ca2+ channel (InsP3 receptor). In porcine cerebellar microsomes, the extent of InsP3-induced Ca2+ release (IICR) is almost completely inhibited by 50 microM curcumin (IC50 = 10 microM). As the extent of IICR cannot be restored back to control levels by the addition of excess InsP3 and since it has little effect on [3H]InsP3 binding to cerebellar microsomes, this inhibition is likely to be non-competitive in nature. IICR in cerebellar microsomes is biphasic consisting of a fast and slow component. The rate constants for the two components are both reduced by curcumin to similar extents (by about 70% of control values at 40 microM curcumin). In addition, curcumin also reduces agonist (ATP)-stimulated Ca2+ mobilization from intact HL-60 cells, indicating that curcumin is cell permeant. However, since it also affects intracellular Ca2+ pumps and possibly ryanodine receptors, it may lead to complex Ca2+ transient responses within cells, which may well explain some of its putative therapeutic properties.

The mycotoxin paxilline inhibits the cerebellar inositol 1,4, 5-trisphosphate receptor.

Paxilline, a tremorgenic alkaloid mycotoxin produced by Penicillium paxilline, is a reversible inhibitor of the cerebellar inositol 1,4, 5-trisphophate (InsP(3)) receptor. It inhibits the amount or extent of InsP(3)-induced Ca(2+) release (IICR), at sub-maximal concentrations of InsP(3), in a biphasic manner consistent with two inhibition constants (K(i)'s 6.7 and > or =400 microM). As paxilline does not affect InsP(3) binding to the receptor, it can be considered a non-competitive inhibitor. The fact that IICR is biphasic has been interpreted as there being two populations of InsP(3)-sensitive Ca(2+) stores, which release Ca(2+) in either a fast or slow fashion. This study has shown that the rate constants for Ca(2+) release from both the fast and slow populations are reduced by paxilline (100 microM) by about 70% and 60%, respectively. Detailed analysis of the way different concentrations of paxilline inhibit the rate constants for Ca(2+) release indicates that the population of Ca(2+) stores that contribute to the slower phase of Ca(2+) release is more sensitive to the inhibitory action of paxilline.

No evidence for an enhanced role of endothelial nitric oxide in the maintenance of arterial blood pressure in the IUGR sheep fetus.

The fetus makes a number of physiological adaptations to a restriction of placental substrate supply, including a decrease in body growth and an increase in peripheral vasoconstriction which maintains mean arterial pressure (MAP) and supports a redistribution of cardiac output to key fetal organs. It is not known, however, whether chronic restriction of placental substrate supply results in an enhanced or diminished role for vasodilators such as endothelial nitric oxide in the regulation of MAP. We hypothesised that there is an increased contribution of NO to blood pressure regulation in growth restricted fetuses and that a 2h infusion of a nitric oxide synthase inhibitor, N(omega)-nitro-l-arginine methyl ester (l-NAME) would result in an augmented rise in MAP in chronically hypoxemic, placentally restricted (PR, n=8) fetuses compared to controls (n=6) in late gestation. There was no difference in the increase in fetal MAP and decrease in HR during l-NAME infusion between Control and PR fetuses. In the PR group, fetuses with lower mean gestational PaO(2) had a lower increase in MAP during l-NAME infusion. Thus we have found no evidence for an enhanced role of NO in the maintenance of MAP in the chronically hypoxemic IUGR fetus.

Restriction of placental function alters heart development in the sheep fetus.

Placental insufficiency, resulting in restriction of fetal substrate supply, is a major cause of intrauterine growth restriction (IUGR) and increased neonatal morbidity. Fetal adaptations to placental restriction maintain the growth of key organs, including the heart, but the impact of these adaptations on individual cardiomyocytes is unknown. Placental and hence fetal growth restriction was induced in fetal sheep by removing the majority of caruncles in the ewe before mating (placental restriction, PR). Vascular surgery was performed on 13 control and 11 PR fetuses at 110-125 days of gestation (term: 150 +/- 3 days). PR fetuses with a mean gestational Po(2) < 17 mmHg were defined as hypoxic. At postmortem (<135 or >135 days), fetal hearts were collected, and cardiomyocytes were isolated and fixed. Proliferating cardiomyocytes were counted by immunohistochemistry of Ki67 protein. Cardiomyocytes were stained with methylene blue to visualize the nuclei, and the proportion of mononucleated cells and length and width of cardiomyocytes were measured. PR resulted in chronic fetal hypoxia, IUGR, and elevated plasma cortisol concentrations. Although there was no difference in relative heart weights between control and PR fetuses, there was an increase in the proportion of mononucleated cardiomyocytes in PR fetuses. Whereas mononucleated and binucleated cardiomyocytes were smaller, the relative size of cardiomyocytes when expressed relative to heart weight was larger in PR compared with control fetuses. The increase in the relative proportion of mononucleated cardiomyocytes and the relative sparing of the growth of individual cardiomyocytes in the growth-restricted fetus are adaptations that may have long-term consequences for heart development in postnatal life.

Subtype identification and functional properties of inositol 1,4, 5-trisphosphate receptors in heart and aorta.

One of the major mechanisms by which hormones elevate intracellular Ca(2+)levels is by generating the second messenger inositol 1,4, 5-trisphosphate (InsP(3)), which activates a Ca(2+)channel (InsP(3)receptor) located in the endoplasmic reticulum (ER). This study undertakes to identify the InsP(3)receptor subtypes (isoforms) in heart and aorta and to characterize their functional properties. The InsP(3)receptor isoforms were identified from rat heart and aorta tissues using both reverse-transcriptase polymerase chain reaction (RT-PCR) to assess the presence of mRNA for the different isoforms and immunochemistry using InsP(3)receptor isoform-specific antibodies. Functional studies included ligand binding experiments using [(3)H]InsP(3)and InsP(3)-induced Ca(2+)release studies using Fluo-3 as the Ca(2+)sensing dye. All three isoforms of the InsP(3)receptor were identified using RT-PCR and immunochemical analyses. [(3)H]InsP(3)binding studies using microsomes derived from these tissues showed that heart had a 3-fold lower abundance of InsP(3)receptors than aorta, while both have considerably lower abundance than the well characterized cerebellar microsomes. The affinity of the InsP(3)binding to the receptor was also different in the three tissues. In cerebellum the K(d)was 60 nM, while aorta had a much higher K(d)of 220 nM. Heart microsomes, appeared to show two classes of binding affinity with K(d)s of 150 nM and 60 nM. Furthermore, the effects of free [Ca(2+)] on [(3)H]InsP(3)binding levels were also different for the three tissues. InsP(3)binding to both cerebellar and aorta microsomes decreased by 90% and 60%, respectively, above 30 nM free [Ca(2+)], while InsP(3)binding to heart was relatively insensitive to changes in [Ca(2+)]. At maximal InsP(3)concentrations, aorta microsomes were able to release about 5% of the accumulated Ca(2+), compared to 25% by cerebellar microsomes. Heart microsomes, however, showed only very little InsP(3)-induced Ca(2+)release ( <0.5%). The EC(50)concentration for InsP(3)-induced Ca(2+)release was 1.2 micro M for aorta while that for cerebellum was 0.3 micro M. Known agonists of the cerebellar InsP(3)receptor such as 3-deoxy InsP(3)and adenophostin A were also able to mobilize Ca(2+)from aorta microsomes. In addition, the competitive antagonist heparin and the non-competitive antagonists of the cerebellar InsP(3)receptor, tetracaine and tetrahexylammonium chloride, were also able to block InsP(3)-induced Ca(2+)release from aorta microsomes.