Effects of polypharmacy on the prevalence of adverse drug events resulting in outpatient visits and hospitalization.

A high proportion of hospitalizations is attributable to the prevalence of adverse drug events. This retrospective study included outpatients and inpatients to determine the prevalence of adverse drug events and if polypharmacy increases it. The prevalence, classification, and causality of adverse drug events were assessed based on medical records, laboratory values, and other data. Multivariate analysis (multiple logistic regression analysis) was performed with the presence or absence of adverse drug events at the time of the visit as the dependent variable and items for which the P-value was <0.25 in the univariate analysis as independent variables. The prevalence of adverse drug events was 13.0%, 10.9%, and 16.0% among all patients, the outpatient group, and the inpatient group, respectively. Multivariate analysis showed that polypharmacy (≥5 drugs) significantly increased the risk of adverse drug events in all patients. The prevalence of adverse drug events significantly increased with each additional drug used. We expect that minimizing the number of medications through moderation of the number of prescription drugs and elimination of polypharmacy will reduce the number of outpatient visits and hospitalizations due to adverse drug events.

Involvement of P2 purinoceptors in the regulation of DNA synthesis in the neural retina of chick embryo.

The activation of P2 purinoceptors induces Ca2+ mobilization in the early embryonic chick neural retina. This purinergic Ca2+ response declines parallel with the decrease in mitotic activity during retinal development. To investigate the role of P2 purinoceptors in the regulation of retinal cell proliferation, we studied the effects of the P2 purinoceptor antagonists suramin and pyridoxalphosphate-6-azophenyl-2',4'-disulphonic acid (PPADS), and of the agonist ATP on DNA synthesis in retinal organ cultures from embryonic day 3 (E3) chick. Suramin inhibited [3H]-thymidine incorporation in a dose-dependent manner (IC50: approximately 70 microM). PPADS also reduced [3H]-thymidine incorporation with maximum inhibition of 46% at 100 microM. Exogenous ATP enhanced [3H]-thymidine incorporation in a dose-dependent manner to maximally 200% of control (EC50: approximately 70 microM). In dissociated retinal cultures from E7 chick, both antagonists showed similar inhibitory effects on [3H]-thymidine incorporation without affecting cell viability. In line with these observations, the presence of extracellular ATP was demonstrated both in vitro and in vivo. In the medium of E3 retinal organ cultures, the concentration of ATP increased 25-fold within 1 h of incubation and this concentration was kept for at least 24 h. In the chick amniotic fluid, the ATP concentration was nearly 3 microM at E3 and declined to 0.15 microM at E7. The results indicate that P2 purinoceptors activated by autocrine or paracrine release of ATP are involved in the regulation of DNA synthesis in the neural retina at early embryonic stages.

Ca2+ mobilization and capacitative Ca2+ entry regulate DNA synthesis in cultured chick retinal neuroepithelial cells.

Release of Ca2+ from intracellular Ca2+ stores (Ca2+ mobilization) and capacitative Ca2+ entry have been shown to be inducible in neuroepithelial cells of the early embryonic chick retina. Both types of Ca2+ responses decline parallel with retinal progenitor cell proliferation. To investigate their potential role in the regulation of neuroepithelial cell proliferation, we studied the effects of 2,5-di-tert-butylhydroquinone (DBHQ), an inhibitor of the Ca2+ pump of intracellular Ca2+ stores, and of SK&F 96365, an inhibitor of capacitative Ca2+ entry, on DNA synthesis in retinal organ cultures from embryonic day 3 (E3) chicks and in dissociated cultures from E7 and E9 chick retinae. We demonstrate that both antagonists inhibit [3H]-thymidine incorporation in a dose-dependent manner without affecting cell viability or morphology. The inhibition of [3H]-thymidine incorporation by SK&F 96365 occurred in the same concentration range (IC50: approximately 4 microM) as the blockade of capacitative Ca2+ entry in the E3 retinal organ culture. At a concentration of 5 microM SK&F 96365. DNA synthesis was reduced by 71, 40 and 32% in the E3, E7 and E9 cultures, respectively. Application of DBHQ at concentrations which led to depletion of intracellular Ca2+ stores also inhibited [3H]-thymidine incorporation with IC50 values of 20-30 microM in the different cultures. Our results suggest the involvement of Ca2+ mobilization and capacitative Ca2+ entry in the regulation of DNA synthesis in the developing neural retina.

Regulation of capacitative Ca2+ entry by tyrosine phosphorylation in the neural retina of chick embryo.

Capacitative Ca2+ entry occurs in the neural retina of chick embryo during neurogenesis. We studied the effects of blockers of capacitative Ca2+ entry (SK&F 96365, Zn2+, Ni2+), genistein, a tyrosine kinase inhibitor and vanadate, a protein-phosphotyrosine phosphatase inhibitor in the embryonic chick retina with fura-2 fluorescence measurements. After incubation of the retina in a Ca2+-free solution with or without an inhibitor of Ca2+ pumps of intracellular Ca2+ stores, re-introduction of extracellular Ca2+ caused capacitative Ca2+ entry which was inhibited by SK&F 96365 (10 microM), Zn2+ (1 mM), Ni2+ (5 mM) and genistein (100 microM). On the contrary, vanadate (1 mM) enhanced the Ca2+ entry. These results suggested that tyrosine phosphorylation was involved in the activation of capacitative Ca2+ entry.

Identification and characterization of isoenzymes of cyclic nucleotide phosphodiesterase in human kidney and heart, and the effects of new cardiotonic agents on these isoenzymes.

The present study was done to identify and characterize the isoenzymes of cyclic nucleotide phosphodiesterase (PDE) and to determine their intracellular distribution in human kidney and heart. The in vitro effects of new cardiotonic agents, namely, NSP-805 (4,5-dihydro-5-methyl-6-[4-[(2-methyl-3-oxo-1-cyclopentenyl)amino] phenyl]-3(2H)-pyridazinone), TZC-5665 (6-[4-[2-[3-(5-chloro-2-cyanophenoxy)-2-hydroxypropylamino]- 2 -methylpropylamino]phenyl]-5-methyl-4,5-dihydro-3(2H)-pyridazinone ) and its metabolites, OPC-18790 ((+/-)-6-[3-(3,4-dimethoxybenzylamino)-2 -hydroxypropoxy]-2-(1H)-quinolinone), MS-857 (4-acetyl-1-methyl-7-(4-pyridyl)-5,6,7,8-tetrahydro-3(2H)-isoquinolinone ) and E-1020 (1,2-dihydro-6-methyl-2-oxo-5-(imidazo[1,2-a]pyridin-6-yl)-3-pyridine carbonitrile hydrochloride monohydrate), on these human PDE isoenzymes were also investigated. PDE isoenzymes were separated from cytosolic and particulate fractions of homogenates of human kidney and heart by DEAE-Sepharose chromatography. PDE isoenzymes were identified by their elution characteristics, substrate specificities, sensitivities to regulation by effectors and by the use of isoenzyme-specific inhibitors. In a cytosolic fraction from kidney, Ca2+/calmodulin-dependent PDE (CaM-PDE), cyclic GMP-stimulated PDE (cGS-PDE), cyclic GMP-inhibited PDE (cGI-PDE) and two forms of cyclic AMP-specific PDE (cAMP-PDE) were resolved. One form of cAMP-PDE (cAMP-PDE alpha), which was eluted at a lower ionic strength than cGI-PDE during DEAE-Sepharose chromatography, was newly recognized in human tissues, though the other form (cAMP-PDE beta), which eluted later than cGI-PDE, had been previously isolated.(ABSTRACT TRUNCATED AT 250 WORDS)

Ca2+ responses to ATP via purinoceptors in the early embryonic chick retina.

1. The action of adenosine triphosphate on cytoplasmic Ca2+ concentration ([Ca2+]i) was studied in the retinal cell of early embryonic chicks with fura-2 fluorescence measurements. The fluorescence was measured from the whole neural retina dissected from chick embryos at embryonic day three (E3). 2. Bath application of ATP (> or = 30 microM; EC50, 128 microM) raised [Ca2+]i by the release of Ca2+ from intracellular Ca2+ stores, since the Ca2+ response to ATP occurred even in a Ca(2+)-free medium. 3. The Ca2+ response to ATP was mediated by P2U purinoceptors. An agonist for P2U purinoceptors, uridine triphosphate (UTP), evoked Ca2+ rises more potently (> or = 3 microM; EC50, 24 microM) than ATP. Agonists for P2X purinoceptors, alpha, beta-methylene ATP and beta, gamma-methylene ATP, or an agonist for P2Y purinoceptors, 2-methylthio ATP (500 microM each), caused no Ca2+ response. Suramin (100 microM) and Reactive Blue 2 (50 microM) almost completely blocked the Ca2+, responses to 500 microM ATP and 200 microM UTP. 4. The developmental profile of the Ca2+ response to ATP was studied from E3 to E13. The Ca2+ response to ATP was largest at E3, drastically declined towards E8 and decreased further until E11-13. 5. These results suggest that the Ca2+ mobilization by ATP via P2U purinoceptors is characteristic of early embryonic retinal cells.

Development of glutamate-induced intracellular Ca2+ rise in the embryonic chick retina.

Neurotransmitters affect neuronal development by regulating intracellular Ca2+ concentrations. We studied spatiotemporal pattern of the development of glutamate-induced intracellular Ca2+ rise in the embryonic chick retina, where developmental changes in mitotic activity, cell death, and synapse formation have been well established. Glutamate was bath-applied to the central part of the retina dissected at embryonic day 3 (E3) to E13, and changes in intracellular Ca2+ concentration were measured with Fura-2 fluorescence. The Ca2+ rise to glutamate first appeared at E6, reached a maximum at E9-10, and then declined before the appearance of synaptic structures (E12). Ca2+ rises to kainate (KA) and alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) appeared earlier and were larger in amplitude than those to N-methyl-D-aspartic acid. The KA/AMPA receptor of the E9 chick retina was permeable for Ca2+, suggesting the functional expression of Ca2+-permeable KA/AMPA receptors at the stage of retinal cell death. The Ca2+ rise to glutamate and KA occurred intensely at the inner plexiform layer, the inner part of inner nuclear layer, and the ganglion cell layer, where the cell death occurs. The Ca2+ rise to high K+, in contrast, occurred intensely at the nerve fiber layer and the ganglion cell layer, developing continuously from E3 until E11. Our study shows that the Ca2+ rise to glutamate develops with the decline of the mitotic activity of the retinal cells and is transiently enhanced during the period of cell death in the embryonic chick retina.

Changes of compound action potentials in retrograde axonal degeneration of rat optic nerve.

Electrophysiological recordings of compound action potentials (CAPs) were made from the optic nerve (ON) following unilateral optic tract (OT) transection in adult rats. In response to optic chiasm stimulation CAPs recorded from the ON ipsilateral to the transected OT, i.e., the ON of control side, revealed three positive waves, termed n1, n2, and n3 components, corresponding to fast-, intermediate-, and slow-conducting fibers. Two weeks after OT transection, CAPs recorded from the ON contralateral to the transected OT, i.e., the ON of cut side, were generally smaller in amplitude than those recorded from the ON of control side. The degree of amplitude reduction was different among three components; n3 component was severely reduced and n2 component was moderately reduced, whereas n1 component relatively persisted. These tendencies were more marked in CAPs recorded at 4 weeks after OT transection; n3 component disappeared, whereas n1 and n2 components decreased in amplitude with elongation of latency. Cross sections of the ON after unilateral OT transection were examined electron microscopically, in which significant decrease in fiber density, demyelination, and distorted fibers were verified in the cut side. The present study suggests that slower-conducting ON fibers, that is, smaller-diameter ON fibers are initially and remarkably thrown into retrograde degeneration, while larger-diameter ON fibers are relatively resistant to axotomy.

Capacitative Ca2+ influx in the neural retina of chick embryo.

Depletion of intracellular Ca2+ stores induces a capacitative Ca2+ influx in non-neural cells. It has been unknown whether the capacitative Ca2+ influx occurs in the cells of nervous systems. We found the capacitative Ca2+ influx in the neural retina of early embryonic chick with Fura-2 fluorescence measurements. A Ca(2+)-free medium containing thapsigargin (500 nM), an inhibitor of Ca(2+)-ATPase of intracellular Ca2+ stores, was applied to the neural retina of embryonic day 3 (E3) chick. A rise in intracellular Ca2+ concentration was evoked after the reintroduction of extracellular Ca2+, and this Ca2+ rise was suppressed by Zn2+ (1 mM) and Ni2+ (5 mM). The developmental changes in the Ca2+ rise induced by thapsigargin (250 nM) were studied from E3 to E13. The thapsigargin-induced Ca2+ rise was largest at E3, declined rapidly toward E6, and then decreased gradually until E13, when the Ca2+ rise almost disappeared. This developmental profile correlated with the decline in the mitotic activities of the retinal cells studied by Prada et al. The fluorescence imaging with the vertical slice of the E9 retina showed that the site at which the thapsigargin-induced Ca2+ rise was largest was the most outer layer of the retina, where proliferating cells are located. This spatial distribution and the above developmental profile may suggest that the capacitative Ca2+ influx occurs at the early period of neurogenesis when the cells have mitotic activities.

Lysophosphatidic acid-induced Ca(2+) mobilization in the neural retina of chick embryo.

Lysophosphatidic acid (LPA) plays various roles in the regulation of cell growth as a lipid mediator. We studied the effect of LPA on intracellular Ca(2+) concentration ([Ca2+]i) with Fura-2 in the neural retina of chick embryo during neurogenesis. Bath application of LPA (1-100 microM) to the embryonic day 3 (E3) chick retina caused an increase in [Ca2+](i) in a dose-dependent manner, with an EC(50) value of 9.2 microM. The Ca(2+) rise was also evoked in a Ca(2+)-free medium, suggesting that release of Ca(2+) from intracellular Ca(2+) stores (Ca(2+) mobilization) was induced by LPA. U-73122, a blocker of phospholipase C (PLC), inhibited the Ca(2+) rise to LPA. Pertussis toxin partially inhibited the Ca(2+) rise to LPA, indicating that G(i)/G(o) protein was at least partially involved in the LPA response. The developmental profile of the LPA response was studied from E3 to E13. The Ca(2+) rise to LPA declined drastically from E3 to E7, in parallel with decrease in mitotic activity of retinal progenitor cells. The signal transduction pathway and developmental profile of the Ca(2+) response to LPA were the same as those of the Ca(2+) response to adenosine triphosphate (ATP), which enhances the proliferation of retinal progenitor cells. The coapplication of LPA with ATP resulted in enhancement of Ca(2+) rise in the E3 chick retina. Our results show that LPA induces Ca(2+) mobilization in the embryonic chick retina during neurogenesis.

Two isoforms of cGMP-inhibited cyclic nucleotide phosphodiesterases in human tissues distinguished by their responses to vesnarinone, a new cardiotonic agent.

We examined the inhibitory effects of vesnarinone, a new cardiotonic agent, on human cyclic nucleotide phosphodiesterase (PDE). Vesnarinone selectively inhibited the activity of human cardiac cGMP-inhibited PDE with a Ki value of 8.5 microM. The inhibition of human cardiac cGMP-inhibited PDE by vesnarinone was not competitive but was of the mixed type with respect to cAMP. Although the activities of the cGMP-inhibited PDE from human heart, aorta, platelets, and kidney were inhibited to the same extent by cGMP, enoximone, and cilostazole, vesnarinone inhibited the activities of cardiac and kidney cGMP-inhibited PDE with 10 times greater potency than those of platelet and aorta cGMP-inhibited PDE. These results suggest that there exist, in human tissues, two isoforms of cGMP-inhibited PDE that can be distinguished by reference to the inhibitory effects of vesnarinone.

Inhibitory effect of OP-41483.alpha-CD, a prostacyclin analog, on peripheral vascular lesion models in rats.

The effect of a chemically stable prostacyclin analog, OP-41483 alpha-cyclodextrin clathrate (OP-41483.alpha-CD), on vascular lesions, platelet aggregation and blood pressure were examined and compared with those of prostaglandin E1 alpha-cyclodextrin clathrate (PGE1.CD) in in vivo rat models. 1) In the laurate (1 mg/leg, i.a.)-induced arterial thrombotic model, OP-41483.alpha-CD (1 microgram/kg/min, i.v.) prevented the progression of femoral arterial vascular lesions and enhanced the development of collaterals in the femoral artery. PGE1.CD did not inhibit the progression of vascular damages. 2) In the model of vasoconstriction induced by epinephrine (0.05 mg/tail, s.c.) and ergotamine (2 mg/kg, s.c.), OP-41483.alpha-CD and PGE1.CD, at 1 microgram/kg/min, inhibited the progress of of tail gangrene and lessened the decrease in tail cutaneous blood flow. 3) OP-41483.alpha-CD (1 microgram/kg/min) suppressed the ADP (0.1 mg/kg/min, i.v.)-induced decrease in the number of circulating platelets without affecting the change in blood pressure. In contrast, PGE1.CD (3 micrograms/kg/min) inhibited ADP-induced thrombocytopenia with a decrease in blood pressure. These results indicate that OP-41483.alpha-CD has antiplatelet and cutaneous blood flow improving activities that are greater than its hypotensive effect and may be of therapeutic potential in peripheral vascular diseases.

Receptive-field properties of adult cat's retinal ganglion cells with regenerated axons.

Receptive-field properties of retinal ganglion cells (RGCs) that had regenerated their axons were studied by recording single-unit activity from strands teased from peripheral nerve (PN) grafts apposed to the cut optic nerve in adult cats. Of the 286 visually responsive units recorded from PN grafts in 20 cats, 49.7% were classified, according to their receptive-field properties, as Y-cells, 39.5% as X-cells, 6.6% as W-cells, and 4.2% were unclassified. The predominant representation of Y-cells is consistent with a corresponding morphological study (Watanabe et al. 1993a), which identified alpha-cells as the RGC type with the largest proportion of regenerating axons. Among the X-cells, we only found ON-center types, whereas both ON-center and OFF-center Y-cells were found. As in intact retinas, the receptive-field center sizes of Y-cells and W-cells were larger than those of X-cells at corresponding displacements from the area centralis. Within the 10 degrees surrounding the area centralis, the receptive fields of X-cells with regenerated axons were larger than those in intact retinas, suggesting that some rearrangement of retinal circuitry occurred as a consequence of degeneration and regeneration. Receptive-field center responses of Y-, X-, and W-type units with regenerated axons were similar to those found in intact retinas, but the level of spontaneous activity of Y- and X-type units was, in general, less than that of intact RGCs. Receptive-field surrounds were weak or not detected in more than half of the visually responsive RGCs with regenerated axons.

Functional and morphological restoration of intracranial brachial lesion of the retinocollicular pathway by peripheral nerve autografts in adult hamsters.

Axons of adult mammals can regenerate through peripheral nerve grafts and restore the retinocollicular pathway if lesioned proximal to the retinal ganglion cell somata. Whether the grafting and subsequent reinnervation of the superior colliculus (SC) is possible in distal axotomy in the brain is a question of clinical relevance. We have deafferented the SC of adult hamsters at its brachium thus axotomizing the retinal ganglion cell axons rostral to its synaptic contact with the SC neurons. After unilateral brachium transection, a short segment of the autologous sciatic nerve was grafted to bridge the lesioned site to the SC (n = 28). As controls the brachium was transected and left ungrafted (n = 12). Functional restoration was examined 3 to 75 weeks later in grafted (n = 16) and control (n = 5) animals by recording visual evoked responses from the collicular cells. Prior to recording the grafts were visually evaluated and categorized into successfully (n = 8) and unsuccessfully (n = 8) grafted groups. To diffuse flash stimuli applied to the contralateral eye, visual evoked field potentials were recorded from all successfully grafted, but not in unsuccessfully grafted (with the exception of one animal) nor control animals. Unitary spike responses to diffuse flash stimuli were recorded exclusively from three successfully grafted animals. Morphological reinnervation was examined in the remaining grafted (n = 12) and control (n = 7) animals by anterogradely labeling the regenerating retinal axons with WGA-HRP. Axons in the grafts and their terminals in the superficial layers of the SC were clearly labeled in 8 of the grafted and none of the controls. From these results we conclude that the brachium of the SC is conducive to axonal regeneration and the peripheral nerve graft is indeed effective in restoring distally axotomized visual pathway in adult mammals.

Conformational study of a highly specific CXCR4 inhibitor, T140, disclosing the close proximity of its intrinsic pharmacophores associated with strong anti-HIV activity.

We report the solution structure of T140, a truncated polyphemusin peptide analogue that efficiently inhibits infection of target cells by T-cell line-tropic strains of HIV-1 through its specific binding to a chemokine receptor, CXCR4. Nuclear magnetic resonance analysis and molecular dynamic calculations revealed that T140 has a rigidly structured conformation constituted by an antiparallel beta-sheet and a type II' beta-turn. A protuberance is formed on one side of the beta-sheet by the side-chain functional groups of the three amino acid residues (L-3-(2-naphthyl)alanine, Tyr5 and Arg14), each of which is indispensable for strong anti-HIV activity. These findings provide a rationale to dissect the structural basis for the ability of this compound to block the interaction between CXCR4 and envelope glycoproteins from T-tropic strains of HIV-1.