Historic emergence, impact and current status of shrimp pathogens in the Americas.

Shrimp farming in the Americas began to develop in the late 1970s into a significant industry. In its first decade of development, the technology used was simple and postlarvae (PLs) produced from wild adults and wild caught PLs were used for stocking farms. Prior to 1990, there were no World Animal Health Organization (OIE) listed diseases, but that changed rapidly commensurate with the phenomenal growth of the global shrimp farming industry. There was relatively little international trade of live or frozen commodity shrimp between Asia and the Americas in those early years, and with a few exceptions, most of the diseases known before 1980 were due to disease agents that were opportunistic or part of the shrimps' local environment. Tetrahedral baculovirosis, caused by Baculovirus penaei (BP), and necrotizing hepatopancreatitis (NHP) and its bacterial agent Hepatobacterium penaei, were among the "American" diseases that eventually became OIE listed and have not become established outside of the Americas. As the industry grew after 1980, a number of new diseases that soon became OIE listed, emerged in the Americas or were introduced from Asia. Spherical baculovirus, caused by MBV, although discovered in the Americas in imported live Penaeus monodon, was subsequently found to be common in wild and farmed Asian, Australian and African penaeids. Infectious hypodermal and hematopoietic necrosis virus (IHHNV) was introduced from the Philippines in the mid 1970s with live P. monodon and was eventually found throughout the Americas and subsequently in much of the shrimp farming industry in the eastern hemisphere. Taura syndrome emerged in Penaeus vannamei farms in 1991-1992 in Ecuador and was transferred to SE Asia with live shrimp by 1999 where it also caused severe losses. White Spot Disease (WSD) caused by White spot syndrome virus (WSSV) emerged in East Asia in ∼1992, and spread throughout most of the Asian shrimp farming industry by 1994. By 1995, WSSV reached the eastern USA via frozen commodity products and it reached the main shrimp farming countries of the Americas located on the Pacific side of the continents by the same mechanism in 1999. As is the case in Asia, WSD is the dominant disease problem of farmed shrimp in the Americas. The most recent disease to emerge in the Americas was infectious myonecrosis caused by IMN virus. As had happened before, within 3years of its discovery, the disease had been transferred to SE Asia with live P. vannamei, and because of its impact on the industry and potential for further spread in was listed by the OIE in 2005. Despite the huge negative impact of disease on the shrimp farming industry in the Americas, the industry has continued to grow and mature into a more sustainable industry. In marked contrast to 15-20years ago when PLs produced from wild adults and wild PLs were used to stock farms in the Americas, the industry now relies on domesticated lines of broodstock that have undergone selection for desirable characteristics including disease resistance.

Application of molecular diagnostic methods to penaeid shrimp diseases: advances of the past 10 years for control of viral diseases in farmed shrimp.

The most important diseases of farmed penaeid shrimp have infectious aetiologies. Among these are diseases with viral, rickettsial, bacterial, fungal and parasitic aetiologies. Diagnostic methods for these pathogens include the traditional methods of gross pathology, histopathology, classical microbiology, animal bioassay, antibody-based methods, and molecular methods using DNA probes and DNA amplification. While methods using clinical chemistry and tissue culture are standard methods in veterinary and human diagnostic laboratories, the former has not been routinely applied to the diagnosis of penaeid shrimp diseases and the latter has yet to be developed, despite considerable research and development efforts that have spanned the past 40 years. No continuous shrimp cell lines, or lines from other crustaceans, have been developed. Hence, when molecular methods began to be routinely applied to the diagnosis of infectious diseases in humans and domestic animals in the mid- to late 1980s, the technology was applied to the diagnosis of certain important diseases of penaeid shrimp for which only classical diagnostic methods were previously available. A DNA hybridization assay for the parvovirus IHHNV was the first molecular test developed for a shrimp disease. This was followed within a year by the first PCR test for MBV, an important baculovirus disease of shrimp. Today, shrimp disease diagnostic laboratories routinely use molecular tests for diagnostic and surveillance purposes for most of the important penaeid shrimp diseases.

Identification of genomic variations among geographic isolates of white spot syndrome virus using restriction analysis and Southern blot hybridization.

White spot syndrome virus (WSSV) is widely distributed in most of the Asian countries where penaeid shrimp are cultured, as well as in some regions of the USA. Six geographic isolates of WSSV-1 each from penaeid shrimp from China, India, Thailand, and the US states of Texas and South Carolina, and 1 isolated from crayfish at the National Zoological Park in Washington, DC-were compared by combining the methods of restriction analysis and Southern blot hybridization. DNA was extracted from purified viruses and then digested with selected endonucleases: AccI, BglII, ClaI, BamHI, EcoRI, HindII, HaeI, SacI and XhoI. The blots were detected with digoxigenin-11-dUTP-labeled WSSV genomic probes: LN4, C42 and A6. No distinctive differences among the 5 WSSV isolates from penaeid shrimp were detected; however, differences in the WSSV isolate from crayfish were observed. A 2.8 kb DNA fragment originating from the crayfish isolate and encompassing the LN4 region was subcloned into pBluescript and sequenced for comparison with the LN4 fragment from the Thailand WSSV isolate. The results indicate that some genomic components of WSSV from different geographic regions share a high degree of homology. This method can be used to distinguish between the WSSV isolate from crayfish and the WSSV isolates from penaeid shrimp.

Reverse transcription polymerase chain reaction (RT-PCR) used for the detection of Taura syndrome virus (TSV) in experimentally infected shrimp.

Taura Syndrome Virus (TSV) has adversely affected the shrimp culture industries of the Americas. First recognized in 1992, this viral agent has spread throughout the shrimp growing regions of South and Central America to become established in North America in the short span of 5 yr. Diagnostic methods for TSV include histopathology, bioassay using susceptible Penaeus vannamei as the indicator species and in situ hybridization with TSV specific complimentary DNA (cDNA) gene probes. An additional method for detecting TSV is through the use of reverse transcription polymerase chain reaction (RT-PCR). Two oligonucleotide primers were selected using the sequence information from a cloned cDNA segment of the TSV genome. The primers, designated 9195 and 9992, used in the RT-PCR procedure amplify a 231 base pair (bp) fragment of the cDNA. Using the RT-PCR technique, TSV has been detected in the hemolymph of P. stylirostris and P. vannamei with experimentally induced TSV infections.

Risk of spread of penaeid shrimp viruses in the Americas by the international movement of live and frozen shrimp.

Within the past decade, viral diseases have emerged as serious economic impediments to successful shrimp farming in many of the shrimp-farming countries of the world. In the western hemisphere, the viral agents of Taura syndrome (TS) and infectious hypodermal and haematopoietic necrosis have caused serious disease epizootics throughout the shrimp-growing regions of the Americas and Hawaii, while in Asia the viral agents of white spot syndrome (WSS) and yellow head (YH) have caused pandemics with catastrophic losses. The international transfer of live shrimp for aquaculture purposes is an obvious mechanism by which the viruses have spread within and between regions in which they have occurred. Shrimp-eating gulls, other seabirds and aquatic insects may also be factors in the spread of shrimp viruses between and within regions. Another potentially important mechanism for the international spread of these pathogens is the trade in frozen commodity shrimp, which may contain viruses exotic to the importing countries. The viral agents of WSS, YH and TS have been found, and demonstrated to be infectious, in frozen shrimp imported into the United States market. Mechanisms identified for the potential transfer of virus in imported frozen products to domestic populations of cultured or wild penaeid shrimp stocks include: the release of untreated liquid or solid wastes from shrimp importing and processing plants directly into coastal waters, improper disposal of solid waste from shrimp importing and processing plants in landfills so that the waste is accessible to gulls and other seabirds, and the use of imported shrimp as bait by sports fishermen.

Development of a non-radioactive gene probe by PCR for detection of white spot syndrome virus (WSSV).

Combining primers created from the sequence information of two baculo-like viruses of penaeid shrimp, Baculovirus penaei (BP) and Monodon baculovirus (MBV), produced a 750 bp band on a 0.8% agarose gel using White Spot Syndrome Virus (WSSV), from Penaeus monodon, as the DNA template. The PCR fragment was ligated to a plasmid vector, (pGEM-T) and transformed, creating a 3.7 Kbp clone. The DNA insert was sequenced, and the original primer pair was located. Using restriction enzymes, the insert was isolated, excised and non-radioactively labeled. This cloned labeled fragment was tested by in situ hybridization for specificity and reactivity with BP, MBV and WSSV-infected shrimp tissues. The major advantage of this novel method of gene probe development is that no DNA sequence information of the targeted infectious agent needed to be known or available. In addition, tedious viral isolation and purification was circumvented. In this study, knowledge of the possible viral strain was important in limiting the PCR primer pairs investigated. The use of arbitrary primers designed for PCR assays from two other possibly related shrimp viruses, increased the likelihood that a generated PCR product would be specific for WSSV.

Chimeric M1/M2 muscarinic receptors: correlation of ligand selectivity and functional coupling with structural modifications.

Chimeric M1/M2 receptors were expressed in murine fibroblasts (B82) transfected with recombinant m1/m2 receptor genes. The binding affinities of a number of muscarinic antagonists and the agonist carbachol for these chimeric receptors were compared with the ligands' affinities for the M1 and M2 receptors expressed in the B82 cells. The tricyclic compounds, namely pirenzepine (PZ), 11-([2-[(diethylamino)methyl]-1-piperidinyl]acetyl)-5,11- dihydro-6H-pyrido-[2,3-6][1,4]benzodiazepine-6-one (AF-DX 116) and himbacine, shared a binding site between transmembrane domains VI and VII. However, the selective interaction of pirenzepine with M1 and AF-DX 116 and himbacine with M2 involved different structural regions. The high-affinity binding for 4-diphenylacetoxy-N- methylpiperidine and hexahydrosiladifenidol was confined to within loop o2 and transmembrane domains V and VI, which were clearly distinguishable from those of the tricyclic compounds. These results support the hypothesis that the ligands' stereochemical features are critical in their optimal alignment within the ligand binding pocket. The cytoplasmic i3 loop modulated the binding of carbachol such that receptors which contained the i3 domain from the M2 receptor exhibited a single high-affinity state, whereas those with the i3 domain from the M1 receptor had an additional low-affinity state for the agonist. The i3 regions are essential for the differential functional coupling of the M1 and M2 receptors to second messenger systems; however, additional upstream regions seem to be essential for a potent and efficacious activation of phospholipase C by the M1 receptor. This study provides new insight into the molecular basis of ligand selectivity.

A minority of muscarinic receptors mediate rabbit tracheal smooth muscle contraction.

To enhance our understanding of cholinergic mechanisms and muscarinic receptors in bronchoconstriction, we have characterized the muscarinic receptor subtypes in rabbit tracheal smooth muscle using radioligand binding and functional assays. The Kd for [3H]quinuclidinyl benzilate ([3H](-)QNB) binding determined from saturation isotherms was 12.6 x/divided by 1.1 pM (geometric mean x/divided by SEM), and the Bmax was 269 +/- 7 fmol/mg protein (arithmetic mean +/- SEM). Competitive inhibition studies with the muscarinic antagonists pirenzepine (PZ), 11[[2-[(diethylamino)-methyl]1-piperidinyl]acetyl]-5,11-dihydro-6H- pyrido[2,3-b][1,4]benzodiazepine-6-one (AF-DX116), 4-diphenylacetoxy-N-methylpiperidine methobromide (4-DAMP), and hexahydrosiladifenidol (HHSiD) demonstrated heterogeneity of muscarinic receptor subtypes in rabbit tracheal smooth muscle. PZ bound with low affinity to a single receptor site, indicative of an absence of M1 receptors. AF-DX116 (M2 selective) bound with high affinity to approximately 83% of muscarinic binding sites, and 4-DAMP and HHSiD (M3 antagonists) bound with high affinity to approximately 24 and 28% of muscarinic binding sites, respectively. Additionally, direct binding studies with [3H]4-DAMP demonstrated high-affinity binding with 23% of muscarinic binding sites. Thus, the majority of muscarinic receptors in rabbit tracheal smooth muscle bound with high affinity to an M2-selective antagonist, and the remaining receptor sites bound with high affinity to M3 antagonists. The inhibitory effects of atropine, PZ, AF-DX116, and 4-DAMP on methacholine-induced contraction of rabbit tracheal rings were compared. 4-DAMP was a potent inhibitor of methacholine-induced contraction, but PZ and AF-DX116 demonstrated low potency.(ABSTRACT TRUNCATED AT 250 WORDS)

Differential modulation by [D-Pen2, D-Pen5]enkephalin and dynorphin A-(1-17) of the inhibitory bladder motility effects of selected mu agonists in vivo.

The possibility that the delta agonist, [D-Pen2, D-Pen5]enkephalin (DPDPE) and the putative endogenous kappa agonist, dynorphin A-(1-17) could differentially modulate the effects of a group of chemically diverse mu agonists was evaluated using inhibition of volume-induced contractions of the rat urinary bladder as a model of central nervous system opioid receptor function in vivo. Intracerebroventricular administration of equieffective doses of the mu agonists [D-Ala2, NMPhe4, Gly-ol]enkephalin (DAMGO), [N-MePhe3, D-Pro4]enkephalin (PL017), morphine, normorphine, sufentanil, etorphine, phenazocine, meperidine and methadone inhibited spontaneous bladder contractions for approximately 20 to 30 min. Low doses of DPDPE or dynorphin A-(1-17) failed to affect spontaneous bladder contractions; higher doses of DPDPE (greater than 15.5 nmol) and dynorphin A-(1-17) (i.e., greater than 3.7 nmol), inhibited bladder contractions. When coadministered i.c.v., DPDPE displaced the morphine dose-response line to the left and also potentiated the effects of normorphine and etorphine. In contrast, DPDPE failed to alter the actions of equieffective doses of DAGO, PL017, meperidine, methadone, phenazocine or sufentanil. The potentiation of the effects of morphine by DPDPE were prevented by i.c.v. coadministration of the delta antagonist, ICI 174,864 (N,N-diallyl-Tyr-Aib-Aib-Phe-Leu-OH); at the dose tested, the delta antagonist had no agonist effects alone and did not antagonize the effects of morphine directly. Furthermore, the agonist effects of morphine were potentiated by several different doses of DPDPE. Administration of i.c.v. dynorphin A-(1-17) produced a rightward displacement of the morphine dose-response line and also antagonized the effects of normorphine.(ABSTRACT TRUNCATED AT 250 WORDS)

U50,488H differentially antagonizes the bladder effects of mu agonists at spinal sites.

The mu antagonist property of the kappa agonist U50,488H was studied at the spinal level, using motility of the rat urinary bladder as an endpoint in vivo. Intrathecal (i.th.) administration of the mu agonists [D-Ala2,NMePhe4,Gly-ol]enkephalin (DAGO), [N-MePhe3,D-Pro4]enkephalin (PL017), morphine and normorphine, as well as the delta agonist [D-Pen2,D-Pen5]enkephalin (DPDPE), resulted in an equieffective inhibition of volume-initiated contractions of the urinary bladder. In contrast, i.th. administration of U50,488H, a highly selective kappa agonist, had no effect on bladder motility. Pretreatment of rats with i.th. U50,488H prior to agonist administration, blocked the suppression of spontaneous bladder activity induced by equieffective i.th. does of morphine and normorphine, but failed to alter the inhibitory effect of the mu agonists DAGO and PL017, or that of the delta agonist DPDPE. The finding that U50,488H differentially antagonized the identical bladder effects of several mu agonists suggests the presence of mu receptor subtypes (mu isoreceptors) in the rat spinal cord, which may be involved in the regulation of bladder function.

Mu antagonist properties of kappa agonists in a model of rat urinary bladder motility in vivo.

The possibility that the kappa agonists, U50,488H, ethylketazocine and tifluadom, might act as opioid antagonists was studied using the inhibition of the anesthetized rat micturition reflex in vivo as a pharmacological endpoint. Intracerebroventricular administration of equieffective doses of the mu agonists [D-Ala2, NMePhe4, Gly-ol]enkephalin (0.01 nmol), [N-MePhe3, D-Pro4]enkephalin (0.03 nmol), morphine (0.08 nmol), normorphine (0.3 nmol), sufentanil (0.002 nmol), etorphine (0.004 nmol), phenazocine (17 nmol) and meperidine (176 nmol) inhibited spontaneous bladder contractions for a duration of approximately 20 to 30 min. Similarly, i.c.v. administration of the delta-selective agonist (D-Pen2, D-Pen5]enkephalin (15 nmol) inhibited the micturition reflex for approximately the same duration. The kappa agonists U50,488H (22 nmol), ethylketazocine (3 nmol) and tifluadom (3 nmol) did not alter bladder activity after i.c.v. administration. Higher doses of ethylketazocine (10 nmol) or tifluadom (20 nmol), but not U50,488H, produced consistent suppression of bladder contractions. Pretreatment of rats (-15 min, i.c.v.) with doses of U50,488H, ethylketazocine or tifluadom which did not produce an agonist effect consistently blocked the inhibitory actions of the mu agonists morphine and normorphine on bladder motility, but failed to antagonize the similar actions of the mu agonists [D-Ala2, NMePhe4, Gly-ol]enkephalin, [N-MePhe3, D-Pro4]enkephalin, phenazocine, meperidine or those of the delta agonist [D-Pen2, D-Pen5]enkephalin. Centrally initiated bladder effects of the mu agonists etorphine and sufentanil were antagonized by U50,488H but unaffected by ethylketazocine or tifluadom. In addition, administration of U50,488H (i.c.v.) during a morphine-induced bladder shutdown resulted in either an immediate recovery of bladder activity or a shortened duration of action.(ABSTRACT TRUNCATED AT 250 WORDS)

Mu and delta opioid ligands inhibit reflex contractions of the urinary bladder in the rat by different central mechanisms.

The supraspinal and spinal mechanisms of opioid-induced inhibition of reflex contractions of the urinary bladder were studied in female rats, anesthetized with urethane. A variety of central manipulations was made to distinguish the effects produced by [D-Ala2-Me-Phe4-Gly(ol)5]-enkephalin (DAGO), a selective mu-opioid receptor ligand, from those of the delta ligand [2-D-penicillamine, 5-L-penicillamine]-enkephalin (DPLPE), administered by either intracerebroventricular (i.c.v.) or by spinal intrathecal (i.t.) injection. The effect of intraventricular but not of intrathecal administration of DPLPE was abolished 4-5 hr after the systemic administration of reserpine (5 mg/kg, i.p.). Reserpine did not modify the actions of DAGO, given by either route. Pretreatment with 5,7-dihydroxytryptamine (5,7-DHT, 200 micrograms, i.c.v.) attenuated the effect of DPLPE given intraventricularly but not when given intrathecally, measured 7 days later. The effect produced by DPLPE given by either route was unchanged by pretreatment with 6-hydroxydopamine (6-OHDA, 150 micrograms i.c.v.). Neither 5,7-DHT nor 6-OHDA altered the effect of administrations of DAGO. The effect of DPLPE given intraventricularly was attenuated or abolished, in a dose-related and reversible manner, following the administration of naloxone or methysergide intrathecally but not by phentolamine, propranolol or atropine. The effect of DAGO given intraventricularly was antagonised by naloxone but not by any of the other antagonists. These observations suggested that the supraspinally- and spinally-mediated inhibition of reflex contractions of the urinary bladder produced by mu or delta receptor ligands can be dissociated. The supraspinal effect of DPLPE involved a descending serotoninergic, but not adrenergic pathway.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of a novel opioid peptide antagonist on rat bladder motility in vivo.

The agonist, and opioid antagonist, effects of intracerebroventricularly (ICV) given D-Phe-Cys-Tyr-D-Trp-Lys-Thr-Pen-Thr-NH2 (CTP), a cyclic analogue of somatostatin octapeptide, were evaluated using the micturition reflex of the anesthetized rat as the endpoint. Antagonist effects were evaluated against equieffective doses of selective mu [D-Ala2,NMPhe4,Gly-ol]enkephalin (DAGO) and delta [D-Pen2,D-Pen5] enkephalin (DPDPE) opioid agonists. At low ICV doses, CTP preferentially antagonized DPDPE rather than DAGO; increasing the dose of CTP further effectively antagonized both mu and delta agonists, while even higher doses showed an agonist effect alone which was not blocked by adrenergic, cholinergic or opioid antagonists. Selective opioid antagonist doses of CTP failed to block the inhibition of the micturition reflex produced by pentobarbital. Possible residual somatostatin like properties of CTP were tested by using somatostatin as a possible antagonist of equieffective doses of DPDPE and DAGO; somatostatin did not antagonize these agonists. Repeated exposure to CTP resulted in the development of acute tolerance to the agonist effect, and also prevented the inhibition of the reflex by high doses of somatostatin, with the converse experiment showing a similar pattern; thus, repeated somatostatin resulted in tolerance and subsequent cross-tolerance to the agonist effects of CTP. In animals tolerant to somatostatin, CTP nevertheless behaved as an opioid antagonist. The present results indicate that CTP possesses opioid antagonist properties in vivo which are pharmacological in nature but nevertheless retains residual somatostatin-like activity at higher doses.

Supraspinal and spinal mechanisms in morphine-induced inhibition of reflex urinary bladder contractions in the rat.

The supraspinal and spinal mechanisms of morphine-induced inhibition of isometrically recorded reflex urinary bladder contractions were studied in rats anesthetized with urethan. Chronic intracerebroventricular administration of 5,7-dihydroxytryptamine (200 micrograms) or 6-hydroxydopamine (150 micrograms), to selectively deplete central serotoninergic and noradrenergic systems, attenuated the intracerebroventricular effect but not the intrathecal effect of morphine. The intracerebroventricular effect of morphine was reversibly attenuated or abolished by an intrathecal injection of the novel delta-receptor antagonist ICI 174,864 (N,N-diallyl-Tyr-Arb-Aib-Phe-Leu-OH: Aib = alpha-aminoisobutyric acid) (1-3 micrograms) and by intrathecal methysergide (4-10 micrograms), phentolamine (5-10 micrograms), and yohimbine (5-10 micrograms) but not by intrathecal propranolol (10 micrograms), atropine (8 micrograms) or saline (2 micrograms) administered at similar molar concentrations and volumes respectively. These observations support the hypothesis that supraspinal and spinal mechanisms involved in morphine-induced inhibition of reflex urinary bladder contractions can be dissociated. The supraspinal actions of morphine were mediated indirectly via descending 5-hydroxytryptamine and noradrenergic pathways which activated specific 5-hydroxytryptamine and alpha-adrenergic but not beta-adrenergic receptor in the spinal cord. In addition, supraspinal morphine indirectly activated a spinal opioid system which could be directly activated by intrathecal morphine. The similarities between these observations and studies of central pathways mediating nociception and opioid analgesia suggest that similar physiological mechanisms control certain somatic and visceral activity.

Naloxonazine and opioid-induced inhibition of reflex urinary bladder contractions.

Spontaneous volume-induced contractions of the urinary bladder were recorded isometrically in urethane-anesthetized rats. Contractions were inhibited by alternate submaximal but equieffective doses of the selective mu and delta-opioid ligands [D-Ala2-Me-Phe4,Gly(ol)5] enkephaline (DAGO) and [2-D-penicillamine, 5-D-penicillamine] enkephalin (DPDPE), respectively, administered by the intracerebroventricular (i.c.v.) or spinal intrathecal (i.t.) route. Naloxonazine, postulated to be an irreversible mu 1-opioid receptor antagonist, administered by the same route, antagonized the effects of both DAGO and DPDPE. The antagonism of the effect of DAGO was reversed 3-4 hr later but that of DPDPE was more prolonged. Recovery of the effect of DPDPE was observed some 24 hr later. A similar pattern of activity against DAGO and DPDPE given intraventricularly or intrathecally was observed following intravenous injection of naloxonazine (10 mg/kg). Also naloxonazine (i.c.v., i.t. or i.v.) antagonized the effect of morphine given intraventricularly or intrathecally, but antagonism was not observed when morphine was retested 3-4 hr and 24 hr later. Naloxonazine increased the frequency of contraction of the bladder after each route of administration. This effect lasted 1-3 hr and was not seen 24 hr later. Systemic administration of naloxone (10 mg/kg, i.v) also increased the frequency of bladder contraction and attenuated or abolished the effect of DAGO given intraventricularly or intrathecally and the delta-receptor agonist [2-D-penicillamine, 5-L-penicillamine] enkephaline (DPLPE).(ABSTRACT TRUNCATED AT 250 WORDS)

Meptazinol: unusual in vivo opioid receptor activity at supraspinal and spinal sites.

Systemic (1-10 mg/kg, s.c.), intracerebroventricular (i.c.v. 20-80 micrograms) and spinal intrathecal (i.t., 5-20 micrograms) administration of meptazinol hydrochloride produced dose-related inhibition of reflex contractions of the urinary bladder, recorded isometrically in urethane-anesthetized rats. The effects of meptazinol were reversed by naloxone administered by the same route. Indeed, this was achieved with intracerebroventricular or intrathecal administration of naloxone (2 micrograms), which also selectively antagonized the mu-receptor ligand [D-Ala2, MePhe4, Gly(ol)5]enkephalin (DAGO). However ICI 174,864 (3 micrograms, i.c.v. or i.t.), a delta-opioid receptor antagonist, did not affect the actions of meptazinol given intracerebroventricularly or intrathecally though it consistently abolished the equieffective actions of a selective delta-receptor ligand (2-D-penicillamine, 5-L-penicillamine) enkephalin (DPLPE). Naloxonazine (5 micrograms, i.c.v. or i.t.), an irreversible mu 1-opioid receptor antagonist, produced prolonged antagonism of the effects of DPLPE and meptazinol. The effects of DPLPE partially or completely recovered by 24 hr, indicating that naloxonazine produced prolonged antagonism of delta-opioid receptors. The effects of maptazinol however only recovered after 72 hr, suggesting that antagonism by naloxonazine of this ligand was irreversible and was mediated through a unique opioid receptor interaction. Subthreshold doses of meptazinol (10 micrograms, i.c.v.; 3 micrograms, i.t.) consistently antagonized the effects of morphine given intracerebroventricularly or intrathecally but not the equieffective doses of DPLPE or DAGO. These observations suggest that meptazinol inhibited reflex contractions of the bladder by supraspinal and spinal mu-opioid receptor activation. Furthermore, its agonistic effect and its antagonistic actions were compatible with interactions at a subpopulation of opioid receptors, possibly mu 1-receptors.(ABSTRACT TRUNCATED AT 250 WORDS)

Neuropeptide Y depresses reflex urinary bladder contractions in rats and modifies central activity of opioid agonists.

The 36 amino acid peptide neuropeptide Y (NPY) has been found distributed in central structures associated with nociception and the actions of opioid analgesics. We therefore studied its central actions on reflex bladder contractions which we have shown to be inhibited by supraspinal and spinal opioid administrations in urethane anesthetized rats. Neuropeptide Y produced a dose related (0.5-2 micrograms per rat) inhibition of bladder contractions following intracerebroventricular (ICV) and spinal intrathecal (IT) administrations. These effects could not be antagonized by naloxone (2 micrograms, ICV or IT) or by ICI 174,864 [N,N-diallyl-Tyr-Aib-Aib-Phe-Leu-OH: Aib = alpha-aminoisobutyric acid] (3 micrograms, ICV or IT). NPY (0.5-1 micrograms) reduced the ICV and IT effects of morphine but potentiated the action of the selective delta-receptor ligand [2-D-penicillamine, 5-L-penicillamine] enkephalin (DPLPE). The effect of the mu-selective opioid ligand [D-Ala2, Me-Phe4, Gly(ol)5] enkephalin (DAGO) were unaffected as were the submaximal ICV and IT actions of noradrenaline. It was concluded that NPY-induced inhibition of bladder activity was not due to a direct opioid receptor interaction. However since NPY consistently changed the activity of opioids (morphine and DPLPE), this suggested a possible physiological role in the regulation of opioid receptors, central neural excitability and thereby visceral activity.

Centrally administered beta-endorphin produces prolonged changes in delta-opioid ligand activity in vivo.

Spontaneous reflex bladder contractions were recorded isometrically in urethane anesthetized rats. Bladder contractions were depressed by intracerebroventricular injections of the mu-opioid receptor agonist [D-Ala2,MePhe4,Gly(ol)5]enkephalin (DAGO) and the delta-agonist [2D-penicillamine,5D-penicillamine]enkephalin (DPDPE) respectively. The effect of DPDPE was selectively antagonized by ICI 174,864 (N,N-diallyl-Tyr-Aib-Aib-Phe-Leu-OH; Aib = alpha-aminoisobutyric acid). However following the administration of beta-endorphin the antagonistic action of ICI 174,864 could no longer be observed. In addition ICI 174,864 exhibited agonistic activity following beta-endorphin and the effects of DPDPE were prolonged in a dose related manner by beta-endorphin. These observations suggest that beta-endorphin may produce complex changes in central delta-opioid receptor activity.