Biomedical subjects

V S Seybold

Publications and source records attributed to V S Seybold.

At least 19 recordsLinked to original sources

PubMed1998

Nociceptin/orphanin FQ binding increases in superficial laminae of the rat spinal cord during persistent peripheral inflammation.

Regulation of nociceptin/orphanin FQ neurotransmission in conjunction with peripheral inflammation and hyperalgesia was explored, using receptor autoradiography. Binding of [3H]nociceptin was quantified in spinal segment L4 of rats at 2, 4 and 8 days following injection of complete Freund's adjuvant (CFA) into one hind-paw. Densitometric analysis of autoradiograms showed that [3H]nociceptin binding increased in medial and lateral laminae I-II bilaterally 4 days following injection of CFA compared to untreated rats; no change in binding occurred in lamina X at the times examined. Biochemical studies confirmed that the specific binding of [3H]nociceptin to sections of rat brain was consistent with the binding characteristics of the nociceptin receptor. These results suggest that spinal nociceptin receptors are upregulated during hyperalgesia. This response may enhance endogenous mechanisms of antinociception to attenuate the hyperalgesia induced by CFA.

Animals↗

PubMed1998

Bradykinin increases the proportion of neonatal rat dorsal root ganglion neurons that respond to capsaicin and protons.

A number of studies have examined bradykinin-induced sensitization of primary afferent neurons to mechanical or thermal stimuli. However, bradykinin-induced sensitization to other chemical stimuli has not been systematically addressed. We used primary cultures of dorsal root ganglion neurons from neonatal rats to determine whether bradykinin alters the responsiveness of individual neurons to capsaicin and protons. An increase in the concentration of free intracellular Ca2+ was used as a measure of a response to capsaicin or low pH. Pretreatment with bradykinin (30 nM) increased the proportion of "intermediate-size" (240-320 microm2) dorsal root ganglion neurons that responded to capsaicin (100 nM) or low pH (6.1). However, among "small-size" (160-239 microm2) neurons, bradykinin increased the proportion of neurons that responded to low pH (6.1) but not to capsaicin (10 or 100 nM). Because treatment with arachidonic acid (10 microM) did not mimic the effect of bradykinin and inhibition of cyclo-oxygenase and lipoxygenase with 5,8,11,14-eicosatetraynoic acid (10 microM) did not inhibit the effect of bradykinin on the response to capsaicin, it is not likely that the bradykinin-induced enhancement of neuronal responsiveness is mediated by arachidonic acid or its metabolites in this model. These results support the hypothesis that bradykinin sensitizes primary afferent neurons to other chemicals such as protons that are present in inflamed tissue, particularly by recruiting additional sensory neurons to respond to a given chemical stimulus. An increase in the number of responsive nociceptors that innervate inflamed tissue would contribute to hyperalgesia via spatial summation on spinal neurons in the pathway for pain. Furthermore, since bradykinin enhanced the responsiveness of small-size neurons that responded to protons but not to capsaicin, these data suggest that bradykinin-induced sensitization to protons and capsaicin occur by different mechanisms.

Animals↗

PubMed1998

Quantitation of neurokinin 1 receptor internalization and recycling in guinea-pig myenteric neurons.

Agonist-induced endocytosis and recycling of G protein-coupled receptors contributes to desensitization and resensitization of the receptors. In this study, we have used fluorescence immunohistochemistry, confocal microscopy and digital image analysis to quantify the proportion of receptor in the cytoplasm and on the surfaces of nerve cells in the guinea-pig ileum. With these methods we examined the dynamics of internalization of the neurokinin 1 receptor in response to agonist, return of receptor to the cell membrane and its capacity to be re-internalized in response to further exposure to agonist. The basal level of neurokinin 1 receptor immunoreactivity in the cytoplasm was 12-15% of total cellular immunoreactivity. Concentration-response relations were generated for neurokinin 1 receptor internalization after incubation of isolated ileum with 10(-11) to 10(-6) M substance P at 4 degrees C and warming to 37 degrees C for 20 min. The threshold concentration for cytoplasmic receptor to exceed baseline was 10(-11) M and the proportion of receptor in the cytoplasm increased with increasing substance P concentration. The effect of two exposures to agonist was studied using 10(-8) M and 10(-6) M substance P. After equilibration with substance P at 4 degrees C for 1 h followed by 20 min at 37 degrees C with no substance P, neurokinin 1 receptor immunoreactivity in the cytoplasm increased significantly from 12% to 36+/-3% for incubation with 10(-8) M and to 64+/-3% for 10(-6) M. When return of receptor to the surface was blocked with monensin (10(-5) M), 90% of the receptor was in the cytoplasm after 1 h at 37 degrees C following exposure to 10(-6) M substance P. After 60 min without substance P and no monensin, receptor in the cytoplasm decreased to 19+/-2% (10(-8) M) and 38+/-4% (10(-6) M). A second period of equilibration with substance P at 4 degrees C for 1 h followed by 20 min at 37 degrees C, without substance P, resulted in a second wave of endocytosis; the fractions of receptor in the cytoplasm were 47+/-2% (10(-8) M) and 70 2% (10(-6) M). These results indicate that most of the receptors on the cell surface are available for internalization and that the receptors that return to the cell surface after endocytosis rapidly regain their ability to bind ligand and undergo endocytosis.

Animals↗

PubMed1997

Relationship of NK3 receptor-immunoreactivity to subpopulations of neurons in rat spinal cord.

The distribution of immunoreactivity to the neurokinin3 receptor (NK3R) was examined in segments C7, T11-12, L1-2, and L4-6 of the rat spinal cord. NK3R immunoreactivity was visualized by using two antisera generated against sequences of amino acids contained in the C-terminal region of the NK3R. NK3R-immunoreactive cells were numerous in the substantia gelatinosa of all spinal segments examined as well as the dorsal commissural nucleus of spinal segments L1-2. Isolated, immunoreactive cells were scattered throughout other regions of the spinal cord. The relationship of NK3R-immunoreactivity with neurons was demonstrated by colocalization with microtubule associated protein 2-immunoreactivity in individual cells. Within neurons, NK3R-immunoreactivity was associated predominately with the plasma membrane of cell bodies and dendrites. Within the substantia gelatinosa, 86% of nitric oxide synthase (NOS)-immunoreactive neurons were also NK3R-immunoreactive. Although NOS-immunoreactive neurons were found throughout all other regions of the spinal cord in the segments examined, these were not NK3R-immunoreactive. When preganglionic sympathetic neurons in spinal segments T11-12 and L1-2 were visualized by intraperitoneal injection of Fluorogold, less than 1% of the Fluorogold-labeled neurons were also immunoreactive for NK3R. The large number of NK3R-immunoreactive neurons in the substantia gelatinosa suggests that some effects of tachykinins on somatosensation may be mediated by NK3R.

Animals↗

PubMed1997

Spinal NK2 receptors contribute to the increased excitability of the nociceptive flexor reflex during persistent peripheral inflammation.

The role of endogenous neurokinin A in changes in the excitability of spinal neurons during adjuvant-induced, peripheral inflammation was examined by determining the effect of a selective NK2 receptor antagonist, GR103537, on the nociceptive flexor reflex in rats. Intrathecal administration of GR103537 (1.4-14 nmol) dose-dependently attenuated the increased activity of the flexor reflex ipsilateral to the inflamed paw. The activity of GR103537 at NK2 receptors was confirmed by blockade of the facilitation of the reflex by neurokinin A but not substance P in normal rats. These results indicate that endogenous neurokinin A increases the excitability of spinal neurons during persistent peripheral inflammation.

Animals↗

PubMed1997

Calcitonin gene-related peptide induces the formation of second messengers in primary cultures of neonatal rat spinal cord.

This study investigated second messengers formed in response to calcitonin gene-related peptide (CGRP) in primary cultures of neonatal rat spinal cord. CGRP increased the level of cAMP above basal levels (50 pmol/mg protein) over a large range of concentrations. The concentration-response curve had an intermediate plateau at 180 pmol cAMP/mg protein in response to 0.01-0.1 nM CGRP and a maximal plateau of 850 pmol cAMP/mg protein at 300 nM CGRP. The biphasic concentration-response curve (EC50S of 0.7 pM and 22 nM) suggests activation of high- and low-affinity receptors for CGRP. Both neurons and nonneuronal cells contributed to the increase in cAMP formation in response to CGRP. The CGRP receptor blocker, CGRP8-37, inhibited the response to both 1 and 100 nM CGRP, providing additional support for the hypothesis that both high- and low-affinity receptors mediate the formation of cAMP. Only a high concentration of CGRP (1 microM) increased the formation of cGMP, and CGRP had no effect on the formation of inositol phosphates at any of the concentrations tested (0.1-1 microM). These results suggest that CGRP-induced responses in the spinal cord are mediated predominately via the formation of cAMP. The observation that both neurons and nonneuronal cells responded to CGRP indicate that this peptide may have multiple actions in the spinal cord.

Animals↗

PubMed1996

Spinal NK1 receptors contribute to the increased excitability of the nociceptive flexor reflex during persistent peripheral inflammation.

Hyperalgesia is a characteristic of inflammation and is mediated, in part, by an increase in the excitability of spinal neurons. Although substance P does not appear to mediate fast synaptic events that underlie nociception in the spinal cord, it may contribute to the hyperalgesia and increased excitability of spinal neurons during inflammation induced by complete Freund's adjuvant. We examined the role of endogenous substance P in changes in the excitability of spinal neurons during adjuvant-induced, peripheral inflammation by determining the effect of a selective NK1 receptor antagonist (RP67580) on the nociceptive flexor reflex in adult rats. Experiments were conducted 2 or 3 days after injection of adjuvant. Animals exhibited moderate thermal hyperalgesia at this time. The flexor reflex was evoked by electrical stimulation of the sural nerve and was recorded in the ipsilateral hamstring muscles. The flexor reflex ipsilateral to the inflamed hindpaw was enhanced approximately two-fold compared to the flexor reflex evoked in untreated animals as determined by the number of potentials and the duration of the reflex. The enhanced reflex in adjuvant-treated animals was most likely due to an increase in the excitability of spinal interneurons because short-latency activity in the hamstring muscles did not differ between untreated animals and adjuvant-treated animals following electrical stimulation of the L5 dorsal root or the nerve innervating the muscle with a stimulus that was 1.3-1.5 times the threshold for excitation of A-fibers. Intrathecal administration of RP67580 (2.3 and 6.8 nmol) attenuated the flexor reflex evoked in adjuvant-treated animals, but had no effect in untreated animals. Intravenous or intraplantar injection of RP67580 (6.8 nmol) did not affect the flexor reflex in adjuvant-treated animals indicating a spinal action of the drug following intrathecal administration. RP68651, the enantiomer of RP67580, was without effect at doses up to 6.8 nmol, indicating that the effects of comparable doses of RP67580 were due to an action of the drug at NK1 receptors. However, intrathecal administration of 23 nmol of both drugs attenuated the reflex in adjuvant-treated and control animals indicating that effects of RP67580 at this dose were not mediated entirely by its action at NK1 receptors. Overall, these data suggest that endogenous substance P has a role in the increased excitability of spinal interneurons observed during persistent inflammation and support the hypothesis that substance P released in the spinal cord contributes to the hyperalgesia that accompanies adjuvant-induced persistent, peripheral inflammation.

Afferent Pathways↗

PubMed1996

Cannabinoid receptor agonists inhibit glutamatergic synaptic transmission in rat hippocampal cultures.

Activation of cannabinoid receptors inhibits voltage-gated Ca2+ channels and activates K+ channels, reminiscent of other G-protein-coupled signaling pathways that produce presynaptic inhibition. We tested cannabinoid receptor agonists for effects on excitatory neurotransmission between cultured rat hippocampal neurons. Reducing the extracellular Mg2+ concentration to 0.1 mM elicited repetitive, transient increases in intracellular Ca2+ concentration ([Ca2+]i spikes) that resulted from bursts of action potentials, as measured by combined whole-cell current clamp and indo-1-based microfluorimetry. Pharmacological characterization indicated that the [Ca2+]i spikes required glutamatergic synaptic transmission. Cannabinoid receptor ligands inhibited stereoselectively the frequency of [Ca2+]i spiking in the rank order of potency: CP 54,939 > CP 55,940 > Win 55,212-2 > anandamide, with EC50 values of 0.36, 1.2, 2.7, and 71 nM, respectively. CP 55,940 was potent, but not efficacious, and reversed the inhibition produced by Win 55,212-2, indicating that it is a partial agonist. Inhibition of [Ca2+]i spiking by Win 55,212-2 was prevented by treatment of cultures with active, but not heat-treated, pertussis toxin. Win 55,212-2 (100 nM) inhibited stereoselectively CNQX-sensitive excitatory postsynaptic currents (EPSCs) elicited by presynaptic stimulation with an extracellular electrode, but did not affect the presynaptic action potential or currents elicited by direct application of kainate. Consistent with a presynaptic site of action, Win 55,212-2 increased both the number of response failures and the coefficient of variation of the evoked EPSCs. In contrast, cannabimimetics did not affect bicuculline-sensitive inhibitory postsynaptic currents. Thus, activation of cannabinoid receptors inhibits the presynaptic release of glutamate via an inhibitory G-protein.

6-Cyano-7-nitroquinoxaline-2,3-dione↗

PubMed1996

Prostaglandin E2 increases the proportion of neonatal rat dorsal root ganglion neurons that respond to bradykinin.

Prostaglandins sensitize some nociceptors to noxious mechanical, thermal and chemical stimuli; however, not all nociceptors are sensitized by prostaglandins. We used cultures of dorsal root ganglion neurons from neonatal rats to determine whether prostaglandins differentially alter the responsiveness of populations of neurons to the chemical stimulus bradykinin. Groups of dorsal root ganglion neurons were defined by size of the cell soma and by the presence of immunoreactivity for substance P. An increase in the concentration of free intracellular Ca2+ was used as an indicator of responsiveness to bradykinin. Pretreatment (5 min) with prostaglandin E2 (100 nM) increased the proportion of intermediate-size neurons (somal areas of 240-320 microns2) that responded to 30 nM bradykinin by two-fold but did not alter the proportion of small-size neurons (somal areas of 160-239 microns2) that responded. Pretreatment with prostaglandin E2 had no effect on the maximum increase in free intracellular Ca2+ evoked by 30 nM bradykinin in either population of neurons, defined by size. Although pretreatment with PGE2 did not increase the proportion of intermediate-size neurons that responded to a lower concentration of bradykinin (3 nM), it did increase the concentration of free intracellular Ca2+ evoked by 3 nM bradykinin. Both results were consistent with a leftward shift in the stimulus-response relationship for bradykinin following pretreatment with PGE2. Small- and intermediate-size neurons that responded to bradykinin also differed in their expression of immunoreactivity for substance P. Furthermore, intermediate-size neurons that expressed immunoreactivity for substance P were more likely to respond to bradykinin after treatment with prostaglandin E2. These results support the hypothesis that prostaglandin E2 sensitizes some normally unresponsive primary afferent neurons to chemical stimuli. One population of neurons which becomes responsive to bradykinin after treatment with prostaglandin E2 can be defined based on cell size, and furthermore, these neurons are likely to express substance P. During inflammation, recruitment of primary afferent neurons that are immunoreactive for substance P would enhance the participation of substance P in central mechanisms that contribute to hyperalgesia.

Animals↗

PubMed1995

Changes of opioid binding density in the rat spinal cord following unilateral dorsal rhizotomy.

Mu, delta and kappa opioid receptors in the vertebrate spinal cord mediate the potent antinociceptive effects of opioid agonists administered onto the spinal cord. The present experiments were conducted to determine the effect of unilateral dorsal rhizotomy on mu, delta and kappa spinal opioid binding sites. Measurements of opioid binding were made at 1, 2, 4 or 8 days after rhizotomy and comparisons were made to intact animals. The changes in mu, delta and kappa opioid binding sites were determined by receptor autoradiography using the highly selective radioligands [3H]sufentanil, [3H]DPDPE and [3H]U69593, respectively. Within autoradiograms of each spinal cord, three regions on each side of the spinal cord were targeted for densitometric analysis: laminae I-II (medial), V (lateral) and X. When effects of unilateral rhizotomy within animals were assessed by comparison of the density of binding on the side ipsilateral to the rhizotomy to the contralateral side, decreases in the binding of all three radioligands were observed in laminae I-II on the side of the spinal cord ipsilateral to the rhizotomy at 2-8 days postlesion. A significant reduction in binding was also noted for mu and delta sites in lamina V after 8 days and for delta binding in lamina X at 2 and 4 days on the side ipsilateral to the rhizotomy. However, when densities of binding sites were compared with the corresponding regions in control, it was clear that dorsal rhizotomy resulted in significant changes in opioid binding on both sides of the spinal cord; changes differed for each type of opioid binding site. On the contralateral side of the spinal cord, rhizotomy caused a significant decrease of mu opioid sites 1 day after the lesion and showed partial recovery by day 8. Delta opioid sites were also significantly decreased as early as 1 day postlesion, but did not recover. Kappa opioid sites did not change at 1 day after the rhizotomy but increased on day 2, decreased on day 4 and fully recovered 8 days after rhizotomy. The present results support the hypothesis that a significant proportion of spinal mu, delta and kappa opioid binding sites are present on the central terminations of primary afferents. Finally the present data are the first to report a contralateral effect of the unilateral rhizotomy on spinal opioid binding sites. The contralateral changes in binding were specific to the type of opioid site examined, time after the surgery and region of the spinal cord examined.

Animals↗

PubMed1995

Plasticity in the synthesis and storage of substance P and calcitonin gene-related peptide in primary afferent neurons during peripheral inflammation.

Several indices of peptidergic, primary afferent neural transmission in rat at the level of the lumbar spinal cord exhibited differential changes over time in response to adjuvant-induced inflammation of the hindpaw. The indices were measurements of the production of messenger RNA encoding the precursors for substance P and calcitonin gene-related peptide in dorsal root ganglia, the storage of substance P and calcitonin gene-related peptide in the dorsal spinal cord and the release of the peptides evoked by application of capsaicin to the dorsal spinal cord. A 47% decrease in the content of immunoreactive substance P in the dorsal half of the lumbar spinal cord, as determined by radioimmunoassay, was measured at 6 h following the injection of complete Freund's adjuvant into the hindpaw. Decreased content of immunoreactive SP persisted for four days, but was no longer present at eight days after the adjuvant injection. The content of immunoreactive calcitonin gene-related peptide in the dorsal spinal cord was decreased by 29% at one day following the injection of adjuvant into the rat hindpaw and 43% at two days; the content then increased to a level greater than that of control animals at eight days. The amount of messenger RNA encoding preprotachykinin and prepro-calcitonin gene-related peptide in L4-L6 dorsal root ganglia was determined from northern blot analysis of the total messenger RNA extracted from the dorsal root ganglia. Each species of messenger RNA had increased compared to the control animals at two days following the injection of adjuvant into the rat hindpaws and remained elevated after eight days. Thus, an increase in the messenger RNAs encoding substance P and calcitonin gene-related peptide in the dorsal root ganglia preceeded the recovery of the content of the peptides in the spinal cord. Morphometric studies of calcitonin gene-related peptide-immunoreactive perikarya in the L4 dorsal root ganglia indicated that the increase in messenger RNA occurred in neurons of the size that normally express calcitonin gene-related protein. Radioimmunoassay of the superfusate of the dorsal half of the lumbar spinal cord was used to measure the release of immunoreactive substance P and immunoreactive calcitonin gene-related protein in vitro. Although the basal release of immunoreactive substance P and immunoreactive calcitonin-gene related protein from the dorsal spinal cord was constant throughout the time points examined, changes occurred in the release of peptide evoked by 10 microM capsaicin. The capsaicin-evoked release of immunoreactive substance P was decreased at 6 h and eight days post-injection of adjuvant.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗

PubMed1995

Tachykinins alter inositol phosphate formation, but not cyclic AMP levels, in primary cultures of neonatal rat spinal neurons through activation of neurokinin receptors.

The naturally occurring tachykinins, substance P, neurokinin A and neurokinin B, induce the formation of inositol phosphates or cAMP in a variety of tissues but their effects on neurons have not been resolved. We used primary cultures of neonatal rat spinal cord to determine whether neurokinin receptors mediate changes in these second messengers in spinal neurons. We found that substance P, neurokinin A and neurokinin B induced the formation of inositol phosphates in a concentration-dependent manner with similar potencies (EC50S: 3.6, 5.7 and 21.3 nM, respectively), but at concentrations tested (0.1-1.0 microM) these peptides had no effect on cAMP levels. All three tachykinins induced the formation of inositol phosphates predominately by activation of neurokinin1 receptors. CP-96,345 and WIN 51,708, neurokinin1 receptor antagonists, attenuated the response to substance P, neurokinin A and neurokinin B. GR 103,537, a neurokinin2 receptor antagonist, had no effect on the responses induced by any of the tachykinins. Furthermore, the selective neurokinin1 receptor agonist, GR-73632, induced the formation of inositol phosphates in a concentration-dependent manner, whereas the selective neurokinin2 receptor agonist, GR-64349, generated inositol phosphates only at the highest concentration tested (10 microM). Senktide, a neurokinin3 receptor agonist, did not induce the formation of inositol phosphates at any of the concentrations tested (0.01-10 microM). Inositol phosphate formation appeared to be due to a direct effect of the tachykinins on neuronal neurokinin1 receptors. These results suggest that biological responses in spinal neurons following activation of neurokinin1 receptors are mediated mainly by the hydrolysis of phosphoinositol 4,5-bisphosphate to form inositol 1,4,5-trisphosphate and diacylglycerol. It remains to be determined which of these second messengers mediates the increased neuronal excitability and depolarization that occurs in response to substance P.

Animals↗

PubMed1995

Characterization and regulation of neurokinin1 receptors in primary cultures of rat neonatal spinal neurons.

Neurokinin1 receptors are the primary target of substance P released from neurons during neural transmission, yet little is known regarding the regulation of neurokinin1 receptors on neurons. 125I-Bolton-Hunter-substance P was used in the present studies to determine whether primary cultures of rat neonatal spinal cord express neurokinin1 receptors and, therefore, can be used as a model to explore regulation of neuronal neurokinin1 receptors. 125I-Bolton-Hunter-substance P bound to a single site in neuron-enriched cultures with an affinity of 256 nM, which is comparable to its high affinity binding on adult rat spinal cord. Treatment of neuron-enriched cultures with 10 nM substance P resulted in a 47% decrease in 125I-Bolton-Hunter-substance P binding at 24 h which was due to a decrease in affinity of the receptor. However, at 48 h after treatment with substance P, 125I-Bolton-Hunter-substance P binding increased by 44%. The increase in binding was due to a two-fold increase in receptor density. These changes occurred in the presence of 0.5 microM tetrodotoxin, decreasing the likelihood that the changes in binding were secondary to the release of transmitters by substance P. Furthermore, substance P1-7, a metabolite of substance P that has putative physiological effects, did not alter 125I-Bolton-Hunter-substance P binding. These data provide evidence that neuronal neurokinin1 receptors are under homologous regulation in primary cultures of neonatal rat spinal cord and suggest that a similar mechanism occurs in vivo. Furthermore, the data suggest that a decrease in affinity of the neurokinin1 receptor may contribute, in part, to tachyphylaxis to substance P.

Animals↗

PubMed1995

Plasticity of calcitonin gene related peptide neurotransmission in the spinal cord during peripheral inflammation.

Injection of complete Freund's adjuvant (CFA; 75 microL) into the plantar surface of the hind paw of the rat results in a mild inflammation that lasts for several days and is accompanied by hyperalgesia. Multiple components of calcitonin gene related peptide (CGRP) neurotransmission in the spinal cord are altered during the course of this peripheral inflammation. The content of immunoreactive (i) CGRP in the dorsal horn of the spinal cord, where primary afferent neurons terminate, is significantly decreased within 2 days after injection of CFA but increases to a level greater than that of the control at 8 days. The early decrease in iCGRP in the spinal cord suggests that the release of CGRP from primary afferent neurons is increased during the period of maximal hyperalgesia that accompanies peripheral inflammation. Changes in the mRNA for CGRP suggest that the increase in spinal content of iCGRP is due to an increase in synthesis of the peptide as the level of mRNA for CGRP is increased from 2 to 8 days after injection of CFA. Despite the decrease in the content of iCGRP in the spinal cord, there is no apparent decrease in the amount of iCGRP that can be released from the dorsal spinal cord by capsaicin; in fact, capsaicin-evoked release is increased at 4 days. Measurements of the binding of 125I-labelled CGRP in the dorsal spinal cord indicate that high affinity binding sites for CGRP are downregulated at 4 days after injection of CFA. In total, these data support the hypothesis that the activity of CGRP-containing primary afferent neurons is increased during peripheral inflammation. CGRP released from primary afferent neurons in the spinal cord may contribute to cellular changes that accompany peripheral inflammation.

Animals↗

PubMed1994

Placental transferrin receptor in diabetic pregnancies with increased fetal iron demand.

Augmented fetal hemoglobin synthesis during diabetic pregnancy increases fetal iron demand. To study the effect of increased fetal iron demand on placental transferrin receptor (TR), we utilized a monoclonal antibody to localize placental TR immunoreactivity and 125I-labeled transferrin to study TR binding characteristics in 10 placentas from poorly controlled diabetic mothers with increased fetal iron demand and 10 placentas from nondiabetic mothers. The infants born to the diabetics had higher cord serum C-peptide, erythropoietin, and hemoglobin concentrations, indicating fetal hyperinsulinemia and hypoxia, with augmented erythropoiesis and iron demand. TR immunoreactivity was localized to the syncytiotrophoblast in both groups, was greater in the diabetic group, and was inversely correlated with fetal storage iron (r = -0.75; P < 0.001). Scatchard analysis of 125I transferrin binding data confirmed greater receptor number (Bmax 17.9 +/- 2.2 vs. 12.6 +/- 1.3 pM/mg protein, P = 0.05), but reduced binding affinity [dissociation constant (Kd) 7.6 +/- 0.9 vs. 5.4 +/- 0.4 nM/l, P = 0.03] in the diabetic group. The TR staining intensity, Bmax, and Kd were each correlated with cord C-peptide, suggesting either a primary or secondary role for fetal hyperinsulinemia in TR expression. This study provides in vivo evidence that fetal factors, such as iron demand or hyperinsulinemia, influence regulation of placental TR in humans. The increase in placental syncytiotrophoblastic TR expression associated with reduced cord serum ferritin concentration suggests that the fetus utilizes both increased placental iron transport and mobilization of fetal iron stores to support augmented fetal erythropoiesis.

Erythropoiesis↗

PubMed1993

Autoradiographic evidence for decrease in binding of mu- and delta-opioid receptors after subchronic [D-Ala2,D-Leu5]enkephalin treatment in rats.

Tolerance to the antinociceptive effect of [D-Ala2,D-Leu5]enkephalin (DADLE) developed in Sprague-Dawley rats given the peptide chronically. delta-Opioid receptor binding is significantly reduced in P2 membranes from various brain areas after 1-3 days' treatment. mu-Opioid receptor binding, however, is reduced only in striatum, and only after 5 days. To study this finding further, receptor autoradiography was used to quantify mu- and delta-opioid binding sites in rat brain sections after subchronic DADLE treatment. Autoradiograms were made following equilibrium binding of the highly selective opioid radioligands, [3H]Tyr-D-Ala-Gly-MePhe-Gly-ol ([3H]DAMGO) and [3H][D-Pen2,5]enkephalin ([3H]DPDPE) to brain sections. Computerized grain counting was applied to discrete regions of the autoradiograms corresponding to caudate and interpeduncular nuclei. We found that [3H]DAMGO binding decreased in caudate after 3 days of DADLE treatment and [3H]DPDPE binding decreased in the interpeduncular nucleus, rostral portion, after 1-day DADLE treatment. These autoradiographic changes are consistent with our earlier results for biochemical binding, although we now detected an earlier change in mu-opioid receptor binding, by using autoradiography.

Amino Acid Sequence↗

PubMed1993

Distribution of [3H]quinuclidinyl benzilate, [3H]nicotine, and [125I]alpha-bungarotoxin binding sites in the nucleus tractus solitarii of the cat.

The distribution of muscarinic and nicotinic cholinergic binding sites in the cat nucleus tractus solitarii was studied by the technique of in vitro autoradiography. Using the antagonist [3H]quinuclidinyl benzilate, muscarinic binding sites were differentially located in subdivisions of the nucleus tractus solitarii. The majority of muscarinic binding sites were located predominantly in the caudal half of the nucleus, reaching their greatest amounts at the mid levels of the nucleus tractus solitarii. The medial, dorsolateral, intermediate, and interstitial subdivisions contained the highest densities of quinuclidinyl benzilate binding sites. Nicotinic cholinergic binding sites, using [3H]nicotine and [125I]alpha-bungarotoxin, had unique patterns of distribution. With [3H]nicotine the majority of binding sites were located in rostral levels of the nucleus with very few binding sites present in the caudal half. In contrast, [125I]alpha-bungarotoxin binding sites were present mainly in subdivisions located in the caudal half of the nucleus, i.e., commissural, ventrolateral, dorsolateral, medial, and intermediate subdivisions, and dropped off precipitously at more rostral levels. The differential distribution of [3H]nicotine and [125I]alpha-bungarotoxin suggests the two ligands may be labeling different types of nicotinic binding sites in the nucleus tractus solitarii. The unique distribution of muscarinic and nicotinic cholinergic binding sites in the various subdivisions of the nucleus solitarii suggests that muscarinic and nicotine mechanisms may play an active role in the regulation of the diverse autonomic functions at the level of the nucleus tractus solitarii.

Acetylcholine↗

PubMed1993

Time-dependent changes in Bolton-Hunter-labeled 125I-substance P binding in rat spinal cord following unilateral adjuvant-induced peripheral inflammation.

Time-dependent changes in Bolton-Hunter-labeled 125I-substance P binding occurred in the dorsal horn of the spinal cord following unilateral adjuvant-induced inflammation in the hindpaw of the rat. Inflammation was characterized by measures of edema and hyperalgesia. Edema and hyperalgesia were both present 6 h after induction of inflammation. However, by eight days, hyperalgesia had dissipated while edema persisted. Six hours after the induction of inflammation, widespread decreases in Bolton-Hunter-labeled 125I-substance P binding occurred on both sides of the dorsal horn of spinal level L4 in comparison to the control group. However, by two days, widespread increases in Bolton-Hunter-labeled 125I-substance P binding occurred on both sides of the spinal cord at level L4 compared to the control group. The increase in radioligand binding was primarily due to a 10-fold increase in affinity of neurokinin-1 receptors for substance P. At later time-points of four and eight days, Bolton-Hunter-labeled 125I-substance P binding remained increased only in laminae I/II on the side of the spinal cord ipsilateral to inflammation. The changes in Bolton-Hunter-labeled 125I-substance P binding suggest that alterations in substance P synaptic transmission in the spinal cord may contribute to the increased excitability of spinal neurons that accompanies adjuvant-induced peripheral inflammation.

Animals↗