Biomedical subjects

H Nagase

Publications and source records attributed to H Nagase.

At least 37 records · Page 2Linked to original sources

PubMed2001

Up-regulation of spinal mu-opioid receptor function to activate G-protein by chronic naloxone treatment.

The effects of repeated s.c. administrations of an mu-opioid receptor antagonist naloxone on the G-protein activation induced by mu-opioid receptor agonists [D-Ala(2),N-MePhe(4),Gly-ol(5)]enkephalin (DAMGO), endomorphin-1 and endomorphin-2 in the mouse spinal cord was studied, monitoring guanosine-5'-o-(3-[35S]thio)triphosphate ([35S]GTPgammaS) binding. All mu-opioid receptor agonists concentration-dependently increased the [35S]GTPgammaS binding. The increases of [35S]GTPgammaS binding induced by agonists were significantly enhanced in mice pretreated with naloxone. Under the present condition, chronic treatment with naloxone significantly increased the density of [3H]DAMGO binding sites with an increase in K(d) values in spinal cord membranes, indicating an increase in mu-opioid receptors on the membrane surface. These findings suggest that chronic treatment with an mu-opioid receptor antagonist naloxone leads to the supersensitivity to activate G-protein by mu-opioid receptor agonists with an increase in mu-opioid receptors in membranes of the mouse spinal cord.

Analgesics, Opioid↗

PubMed2001

Lack of the involvement of mu1-opioid receptor subtype on motivational effects induced by the endogenous mu-opioid receptor ligands endomorphin-1 and -2 in the mouse.

The present study was designed to investigate the role of mu-opioid receptor subtypes in the motivational effect of endogenous mu-opioid receptor ligands, endomorphin-1 and -2. In C57BL/6J mice, endomorphin-1 produced a significant place preference, whereas endomorphin-2 exhibited a significant place aversion. These effects were abolished by a mu1/mu2-opioid receptor antagonist beta-funaltrexamine. Under these conditions, both endomorphin-1 and -2 produced their motivational effects in mu1-opioid receptor-deficient CXBK mice, indicating the mu2-opioid receptor involvement. Furthermore, in the lower midbrain including ventral tegmental area, both endomorphin-1 and -2 equally produced dose-related increases in guanosine-5'-o-(3-[35S] thio) triphosphate bindings in C57BL/6J and CXBK mice. These findings indicate that endomorphin-1 and -2 may produce distinct motivational effects via respective mu2-opioid receptor isoforms in the mouse. Furthermore, endomorphin-1 and -2 produced the mu1-resistant G-protein activation in the mouse lower midbtrain.

Analgesics, Opioid↗

PubMed2001

Tumor autocrine motility factor is an angiogenic factor that stimulates endothelial cell motility.

Autocrine motility factor (AMF) is a type of tumor-secreted cytokine which primarily stimulates tumor cell motility via receptor-mediated signaling pathways, and is thought to be connected to tumor progression and metastasis. Using in vivo models, we showed that critical neovascularization responded to a biological amount of AMF. This angiogenic activity was fixed by specific inhibitors against AMF. AMF stimulated in vitro motility of human umbilical vein endothelial cells (HUVECs), inducing the expression of cell surface AMF receptor localizing a single predominant perinuclear pattern closely correlated with its motile ability. AMF also elicited the formation of tube-like structures mimicking angiogenesis when HUVECs were grown in three-dimensional type I collagen gels. We further immunohistochemically detected AMF receptors on the surrounding sites of newborn microvessels. These findings suggest that AMF is a possible tumor progressive angiogenic factor which may act in a paracrine manner for the endothelial cells in the clinical neoplasm, and it will be a new target for antiangiogenic treatment.

Cell Membrane↗

PubMed2001

Tumor autocrine motility factor is an angiogenic factor hat stimulates endothelial cell motility.

Autocrine motility factor (AMF) is a type of tumor-secreted cytokine that primarily stimulates tumor cell motility via receptor-mediated signaling pathways and is thought to be connected to tumor progression and metastasis. Using in vivo models, we showed that critical neovascularization responded to a biological amount of AMF. This angiogenic activity was fixed by specific inhibitors against AMF. AMF stimulated in vitro motility of human umbilical vein endothelial cells (HUVECs), inducing the expression of cell surface AMF receptor localizing a single predominant perinuclear pattern closely correlated with its motile ability. AMF also elicited the formation of tube-like structures mimicking angiogenesis when HUVECs were grown in three-dimensional type I collagen gels. We further immunohistochemically detected AMF receptors on the surrounding sites of newborn microvessels. These findings suggest that AMF is a possible tumor progressive angiogenic factor which may act in a paracrine manner for the endothelial cells in the clinical neoplasm, and it will be a new target for anti-angiogenic treatment.

Angiogenesis Inducing Agents↗

PubMed2001

Involvement of spinal protein kinase Cgamma in the attenuation of opioid mu-receptor-mediated G-protein activation after chronic intrathecal administration of [D-Ala2,N-MePhe4,Gly-Ol(5)]enkephalin.

The present study was designed to investigate the role of a protein kinase C (PKC) isoform in the uncoupling of the mu-opioid receptor from G-proteins after repeated intrathecal injection of a selective mu-receptor agonist, [D-Ala(2),N-MePhe(4),Gly-ol(5)]enkephalin (DAMGO), in the spinal cord of mice. The activation of G-proteins by opioids was measured by monitoring the guanosine-5'-o-(3-[(35)S]thio)triphosphate ([(35)S]GTPgammaS) binding. Mice were injected intrathecally with saline or DAMGO once a day for 1-7 d. At 24 hr after every injection the spinal cord membranes were prepared for the assay. The enhanced [(35)S]GTPgammaS binding by mu-agonists DAMGO, endomorphin-1, or endomorphin-2 was attenuated clearly in spinal cord membranes obtained from mice that were treated intrathecally with DAMGO for 5 and 7 d, but not for 1 or 3 d. By contrast, no change in levels of [(35)S]GTPgammaS binding induced by the delta-receptor agonist SNC-80 or kappa-receptor agonist U-50,488H was noted in membranes obtained from mice that were treated with DAMGO. Concomitant intrathecal administration of a specific PKC inhibitor Ro-32-0432 with DAMGO blocked the attenuation of DAMGO-induced G-protein activation that was caused by chronic DAMGO treatment. Western blotting analysis showed that chronic DAMGO treatment increased the levels of PKCgamma, but not PKCalpha, PKCbetaI, and PKCbetaII isoforms, in spinal cord membranes. Furthermore, mice lacking PKCgamma failed to exhibit the desensitization of the DAMGO-stimulated [(35)S]GTPgammaS binding after repeated DAMGO injection. These findings indicate that repeated intrathecal administration of DAMGO may activate the PKCgamma isoform and in turn cause a desensitization of mu-receptor-mediated G-protein activation in the mouse spinal cord.

Animals↗

PubMed2001

Structural analysis of the alpha(2) integrin I domain/procollagenase-1 (matrix metalloproteinase-1) interaction.

Previous studies have established that ligation of keratinocyte alpha(2)beta(1) integrin by type I collagen induces expression of matrix metalloproteinase-1 (MMP-1) and that MMP-1 activity is required for the alpha(2)beta(1) integrin-dependent migration of primary keratinocytes across collagenous matrices. We now present evidence that MMP-1 binds the alpha(2)beta(1) integrin via the I domain of the alpha(2) integrin subunit. Using an enzyme-linked immunosorbent assay with purified human MMP-1 and recombinant alpha(2) integrin I domain, we showed that the alpha(2) integrin I domain specifically bound in a divalent cation-dependent manner to both the pro and active forms of MMP-1, but not to MMP-3 or MMP-13. Although both the I domain and MMP-1 bind divalent cations, MMP-1 bound, in a divalent cation-dependent manner, to alpha(2) integrin I domains containing metal ion-dependent adhesion sites motif mutations that prevent divalent cation binding to the I domain, demonstrating that the metal ion dependence is a function of MMP-1. Using a series of MMP-1-MMP-3 and MMP-1-MMP-13 chimeras, we determined that both the linker domain and the hemopexin-like domain of MMP-1 were required for optimal binding to the I domain. The alpha(2) integrin/MMP-1 interaction described here extends an emerging paradigm in matrix biology involving anchoring of proteinases to the cell surface to regulate their biological activities.

Antigens, CD↗

PubMed2001

Kinetic analysis of matrix metalloproteinase activity using fluorogenic triple-helical substrates.

Matrix metalloproteinase (MMP) family members are involved in the physiological remodeling of tissues and embryonic development as well as pathological destruction of extracellular matrix components. To study the mechanisms of MMP action on collagenous substrates, we have constructed homotrimeric, fluorogenic triple-helical peptide (THP) models of the MMP-1 cleavage site in type II collagen. The substrates were designed to incorporate the fluorophore/quencher pair of (7-methoxycoumarin-4-yl)acetyl (Mca) and N-2,4-dinitrophenyl (Dnp) in the P(5) and P(5)' positions, respectively. In addition, Arg was incorporated in the P(2)' and P(8)' positions to enhance enzyme activity and improve substrate solubility. The desired sequences were Gly-Pro-Lys(Mca)-Gly-Pro-Gln-Gly approximately Leu-Arg-Gly-Gln-Lys(Dnp)-Gly-Ile/Val-Arg. Two fluorogenic substrates were prepared, one using a covalent branching protocol (fTHP-1) and one using a peptide self-assembly approach (fTHP-3). An analogous single-stranded substrate (fSSP-3) was also synthesized. Both THPs were hydrolyzed by MMP-1 at the Gly approximately Leu bond, analogous to the bond cleaved in the native collagen. The individual kinetic parameters for MMP-1 hydrolysis of fTHP-3 were k(cat) = 0.080 s(-1) and K(M) = 61.2 microM. Subsequent investigations showed fTHP-3 hydrolysis by MMP-2, MMP-3, MMP-13, a C-terminal domain-deleted MMP-1 [MMP-1(Delta(243-450))], and a C-terminal domain-deleted MMP-3 [MMP-3(Delta(248-460))]. The order of k(cat)/K(M) values was MMP-13 > MMP-1 approximately MMP-1(Delta(243-450)) approximately MMP-2 >> MMP-3 approximately MMP-3(Delta(248-460)). Studies on the effect of temperature on fTHP-3 and fSSP-3 hydrolysis by MMP-1 showed that the activation energies between these two substrates differed by 3.4-fold, similar to the difference in activation energies for MMP-1 hydrolysis of type I collagen and gelatin. This indicates that fluorogenic triple-helical substrates mimic the behavior of the native collagen substrate and may be useful for the investigation of collagenase triple-helical activity.

Amino Acid Sequence↗

PubMed2001

Norbinaltorphimine, a selective kappa-opioid receptor antagonist, induces an itch-associated response in mice.

We examined the possibility that scratching induced by norbinaltorphimine, a selective kappa-opioid receptor antagonist, is due to an itch sensation, using compound 48/80 as control pruritogenic agent. When norbinaltorphimine was injected s.c. into the rostral back, mice scratched the skin around the injection site with their hind paws. Although the intensity of the scratching could not be compared because the dose and injection route were different, the character and time course of the scratching behavior induced by compound 48/80 injected i.d. were similar to those with norbinaltorphimine. The scratching behavior induced by norbinaltorphimine was dose-dependently and significantly inhibited by pretreatment with chlorpheniramine. Compound 48/80-induced scratching was also dose-dependently and significantly inhibited by p.o. pretreatment with chlorpheniramine. The scratching behavior induced by norbinaltorphimine was dose-dependently and significantly inhibited by pretreatment with U-50,488H (trans-(+/-)-2-(3,4-dichlorophenyl)-N-methyl-N-[2-(1-pyrrolidinyl)cyclohexyl] acetamide methansulfonate), a kappa-opioid receptor agonist. Unexpectedly, the scratching behavior induced by compound 48/80 was also dose-dependently and significantly reduced by pretreatment with U-50,488H. These results suggest that the injection of norbinaltorphimine into the rostral back of the mouse elicited scratching, which may be an itch-associated response. Furthermore, the scratching behavior produced by norbinaltorphimine may be due in part to the release of histamine followed by antagonism of kappa-opioid receptors.

3,4-Dichloro-N-methyl-N-(2-(1-pyrrolidinyl)-cycloh↗

PubMed2001

The pharmacological profile of delta opioid receptor ligands, (+) and (-) TAN-67 on pain modulation.

We designed the nonpeptidic highly selective delta opioid receptor agonist on the basis of message address concept and the accessory site theory and synthesized (+/-) TAN-67. In spite of highly potent agonistic activity in in vitro assay, (+/-) TAN-67 (racemate) afforded a weak antinociceptive effect in the mouse tail-flick test. This result led us to separate (+/-) TAN-67 to optical pure compounds, (+) and (-) TAN-67. An i.t.-treatment with (-) TAN-67 produced profound antinociceptive effects through specifically acting on delta1 receptors. Unlike (-) TAN-67, i.t.-administered (+) TAN-67 displayed dose-related nociceptive behaviors such as scratching, biting and licking. The effect of (+) TAN-67 was blocked by i.t.-treatment with NTI (delta receptor antagonist) and (-) TAN-67 (delta1 receptor agonist), but not by morphine (mu receptor agonist). The mechanisms involved in spinal pain modulation induced by (+) and (-) TAN-67 were also described.

Analgesics↗

PubMed2001

Mutation W284L of the human delta opioid receptor reveals agonist specific receptor conformations for G protein activation.

Intrinsic activities of different delta opioid agonists were determined in a [35S]GTPgammaS binding assay using cell membranes from Chinese hamster ovary (CHO) cells stably expressing the wild type (hDOR/CHO) or W284L mutant human delta opioid receptor (W284L/CHO). Agonist binding affinities were regulated more robustly by sodium and guanine nucleotide in W284L/CHO than in hDOR/ CHO cell membranes. The W284L mutation selectively reduced the affinity of SNC 80 while having moderate effect ((-) TAN 67) or no effect (DPDPE) on the affinities of other delta selective agonists. The mutation had opposite effects on the intrinsic activities of agonists belonging to different chemical classes. The effects of the mutation on agonist affinities and potencies were independent from its effects on the intrinsic activities of the agonists. Maximal stimulation of [35S]GTPgammaS binding by SNC 80 was 2-fold higher in W284L mutant cell membranes than in wild type hDOR/CHO cell membranes, despite lower receptor expression levels in the W284L/CHO cells. The binding affinity of SNC 80 however, was significantly reduced (15-fold and 30-fold in the absence or presence of sodium+GDP respectively) in W284L/CHO cell membranes relative to wild type hDOR/CHO membranes. Conversely, the Emax of (-)TAN 67 in the [35S]GTPgammaS binding assay was markedly reduced (0.6-fold of that of the wild type) with only a slight (6-fold) reduction in its binding affinity. The affinity and intrinsic activity of DPDPE on the other hand remained unchanged at the W284L mutant hDOR. The mutation had similar effects on the affinities potencies and intrinsic activities of (-)TAN 67 and SB 219825. The results indicate that delta opioid agonists of different chemical classes use specific conformations for G protein activation.

Animals↗

PubMed2001

Role of cholecystokinin in the reduction of endomorphin-2-induced antinociception in diabetic mice.

We examined the role of cholecystokinin in the reduction of endomorphin-2-induced antinociception in diabetic mice. Endomorphin-1 (1-10 microg, i.c.v.) and endomorphin-2 (3-30 microg, i.c.v.) dose dependently inhibited the tail-flick response in non-diabetic and diabetic mice. There was no significant difference between the antinociceptive effect of endomorphin-1 in non-diabetic and diabetic mice. On the other hand, the antinociceptive effect of endomorphin-2 in diabetic mice was significantly less than that in non-diabetic mice. Cholecystokinin octapeptide (CCK-8) dose dependently reduced the antinociceptive effects of endomorphin-1 and endomorphin-2 in non-diabetic mice. However, in diabetic mice, CCK-8 significantly inhibited the antinociceptive effect of endomorphin-1, but not of endomorphin-2. In non-diabetic mice, CI-988 ((R-[R*,R*])-4-([3-1H-indol]-3-yl)-2-methyl-1-oxo-2-([(tricyclo(3.3.1.1)dec-2-yloxy)carbonyl] amino)propylamino-1-phenyl-ethylamino-4-oxybutanoic acid) had no significant effect on either endomorphin-1- or endomorphin-2-induced antinociception. In diabetic mice, while CI-988 had no significant effect on endomorphin-1-induced antinociception, it dose dependently enhanced the antinociceptive effect of endomorphin-2. The results indicated that the reduction of endomorphin-2-induced antinociception in diabetic mice might be due, at least in part, to the activation of CCK(2) receptors.

Analgesics↗

PubMed2001

Application of ion-exchange cartridge clean-up in food analysis IV. Confirmatory assay of benzylpenicillin, phenoxymethylpenicillin, oxacillin, cloxacillin, nafcillin and dicloxacillin, in bovine tissues by liquid chromatography-electrospray ionization tandem mass spectrometry.

A multiresidue analytical method was developed for the confirmation of benzylpenicillin (PCG), phenoxymethylpenicillin (PCV), oxacillin (MPIPC), cloxacillin (MCIPC), nafcillin (NFPC) and dicloxacillin (MDIPC) in bovine tissues using electrospray ionization liquid chromatography-tandem mass spectrometry (LC-ESI-MS-MS) with a product ion scan mode. All penicillins gave [M-H]-, [M-H-CO2]- and [M-H-141]- as the product ion, when [M-H]- was selected as the precursor ion. Combination of an ion-exchange cartridge clean-up and the LC-ESI-MS-MS method can reliably identify all of these penicillins fortified at a concentration of 0.05 mg/kg in bovine tissues, including liver, kidney and muscle. The limits of confirmation satisfy the maximum residue limits for each of the penicillins established by the World Health Organization, US Food and Drug Administration, European Union and Japan.

Animals↗

PubMed2001

The novel kappa-opioid receptor agonist TRK-820 suppresses the rewarding and locomotor-enhancing effects of morphine in mice.

The effects of the novel kappa-opioid receptor agonist TRK-820 on the rewarding and locomotor-enhancing effects of morphine were investigated in mice. Morphine (1-5 mg/kg, s.c.) caused a dose-related preference for the drug-associated place. In contrast, TRK-820 (0.003-0.03 mg/kg, s.c.) did not produce a significant preference for either compartment of the test box. In combination studies, co-injection of TRK-820 (0.01 and 0.03 mg/kg, s.c.) with morphine significantly suppressed the morphine (5 mg/kg, s.c.)-induced place preference, and this effect of TRK-820 was antagonized by pretreatment with nor-BN1 (3 mg/kg, s.c.), a selective kappa-opioid receptor antagonist. TRK-820 also suppressed morphine-induced hyperlocomotion, and this suppression was also blocked by nor-BNI. These results suggest that TRK-820 suppresses the rewarding and locomotor-enhancing effects of morphine through the activation of kappa-opioid receptors. Thus, we propose that TRK-820 may be useful for controlling pain while reducing undesirable side-effects.

Animals↗

PubMed2001

Analysis of benzphetamine and its metabolites in rat urine by liquid chromatography-electrospray ionization mass spectrometry.

An analytical method to identify and determine benzphetamine (BMA) and its five metabolites in urine was developed by liquid chromatography-electrospray ionization mass spectrometry (LC-ESI-MS) using the solid-phase extraction column Bond Elut SCX. Deuterium-labeled compounds, used as internal standards, were separated chromatographically from each corresponding unlabeled compound in the alkaline mobile phase with an alkaline-resistant ODS column. This method was applied to the identification and determination of BMA and its metabolites in rat urine collected after oral administration of BMA. Under the selected ion monitoring mode, the limit of quantitation (signal-to-noise ratio 10) for BMA, N-benzylamphetamine (BAM), p-hydroxybenzphetamine (p-HBMA), p-hydroxy-N-benzylamphetamine (p-HBAM), methamphetamine (MA) and amphetamine (AM) was 700 pg, 300 pg, 500 pg, 1.4 ng, 6 ng and 10 ng in 1 ml of urine, respectively. This analytical method for p-HBMA, structurally closer to the unchanged drug of all the metabolites, was very sensitive, making this a viable metabolite for discriminating the ingestion of BMA longer than the parent drug or other metabolites in rat.

Animals↗

PubMed2001

Epistatic interactions between skin tumor modifier loci in interspecific (spretus/musculus) backcross mice.

The development of cancer is influenced both by exposure to environmental carcinogens and by the host genetic background. Epistatic interactions between genes are important in determining phenotype in plant and animal systems and are likely to be major contributors to cancer susceptibility in humans. Several tumor modifier loci have been identified from studies of mouse models of human cancer, and genetic interactions between modifier loci have been detected by genome scanning using recombinant congenic strains of mice (R. Fijneman et al., Nat. Genet., 14: 465-467, 1996; T. van Wezel et al., Nat. Genet., 14: 468-470, 1996; W. N. Frankel et al., Nat. Genet., 14, 371-373, 1996). We demonstrate here that strong genetic interactions between skin tumor modifier loci can be detected by hierarchical whole genome scanning of a complete interspecific backcross [outbred Mus spretus X Mus musculus (NIH/Ola)]. A locus on chromosome 7 (Skts1) showed a highly significant interaction with Skts5 on chromosome 12 (P < 10(-16)), whereas additional significant interactions were detected between loci on chromosomes 4 and 5, and 16 and 15. Some of these quantitative trait loci and their interactions, in particular the Skts1-Skts5 interaction, were confirmed in two completely independent backcrosses using inbred spretus strains (SEG/Pas and SPRET/Ei) and NIH/Ola. These results, therefore, illustrate the general use of interspecific crosses between Mus musculus and Mus spretus for the detection of strong genetic interactions between tumor modifier genes.

Animals↗

PubMed2001

Enhanced mu-opioid responses in the spinal cord of mice lacking protein kinase Cgamma isoform.

The protein kinase C (PKC)gamma isoform is a major pool of the PKC family in the mammalian spinal cord. PKCgamma is distributed strategically in the superficial layers of the dorsal horn and, thus, may serve as an important biochemical substrate in sensory signal processing including pain. Here we report that mu-opioid receptor-mediated analgesia/antinociception and activation of G-proteins in the spinal cord are enhanced in PKCgamma knockout mice. In contrast, delta- and kappa-opioidergic and ORL-1 receptor-mediated activation of G-proteins in PKCgamma knockout mice was not altered significantly relative to the wild-type mice. Deletion of PKCgamma had no significant effect on the mRNA product of spinal mu-opioid receptors but caused an increase of maximal binding of the mu-opioid receptor agonist [3H][d-Ala(2),N-Me-Phe(4),Gly(5)-ol]enkephalin in spinal cord membranes obtained from PKCgamma knockout mice. These findings suggest that deletion of PKCgamma genes protects the functional mu-opioid receptors from degradation by phosphorylation. More importantly the present data provide direct evidence that PKCgamma constitutes an essential pathway through which phosphorylation of mu-opioid receptors occurs.

Analgesics↗

PubMed2001

TIMP-3 is a potent inhibitor of aggrecanase 1 (ADAM-TS4) and aggrecanase 2 (ADAM-TS5).

The proteoglycan aggrecan is an important major component of cartilage matrix that gives articular cartilage the ability to withstand compression. Increased breakdown of aggrecan is associated with the development of arthritis and is considered to be catalyzed by aggrecanases, members of the ADAM-TS family of metalloproteinases. Four endogenous tissue inhibitors of metalloproteinases (TIMPs) regulate the activities of functional matrix metalloproteinases (MMPs), enzymes that degrade most components of connective tissue, but no endogenous factors responsible for the regulation of aggrecanases have been found. We show here that the N-terminal inhibitory domain of TIMP-3, a member of the TIMP family that has functional properties distinct from other TIMPs, is a strong inhibitor of human aggrecanases 1 and 2, with K(i) values in the subnanomolar range. This truncated inhibitor, which lacks the C-terminal domain that is responsible for interactions with molecules other than active metalloproteinases, is produced at high yield by bacterial expression and folding from inclusion bodies. This provides a starting point for developing a biologically available aggrecanase inhibitor suitable for the treatment of arthritis.

ADAM Proteins↗

PubMed2001

Involvement of central, but not placental corticotropin releasing hormone (CRH) in heat stress induced immunosuppression during pregnancy.

To clarify whether corticotropin releasing hormone (CRH) and beta-endorphin (betaEP) system mediate maternal immunosuppression in pregnant rats exposed to heat through central or placental pathway, we examined the effects of intravenous (iv) (100 or 500 microg) or intracerebroventricular (icv) (5 microg) administration of CRH receptor antagonist alpha-helical CRH (9-41) on splenic natural killer cell activity (NKCA) as well as betaEP in blood, pituitary lobes, and placenta in pregnant rats at 15 to 16 days gestation. Two-way ANOVA revealed that heat reduced NKCA and elevated blood and pituitary betaEP but did not change placental betaEP. Iv administered 500 microg and icv administered alpha-helical CRH reversed the reduced NKCA and the elevated pituitary betaEP, while iv administration of 100 microg alpha-helical CRH did not. The increased blood betaEP was reversed by iv 100 and 500 microg alpha-helical CRH and icv administration. Both iv and icv administrations reduced placental betaEP independent of heat exposure. Thus, the response of placental betaEP to iv administration of alpha-helical CRH seemed to be stronger than that of pituitary betaEP. These results indicate that alpha-helical CRH which acts on pituitary betaEP antagonizes heat-induced immunosuppression during pregnancy, suggesting that immunosuppression produced by heat stress during pregnancy is mediated by the central CRH system. The placental CRH-betaEP system seems unlikely to be involved in the immunosuppression. Physiologic roles of placental CRH and opioid system should be clarified by future in vitro experiments using placenta specimen including placental immunocyte.

Animals↗