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R M Kream

Publications and source records attributed to R M Kream.

At least 37 records · Page 2Linked to original sources
PubMed1994

Expression of substance P and its precursor forms in vagal, tracheal, and lung tissues of the guinea pig.

Steady-state levels of the prototypic tachykinin neuropeptide substance P (SP) and its major precursor form substance P-glycine (SP-G) were detected and authenticated in guinea pig vagal and respiratory tissues by radioimmunoassay (RIA), combined high-performance liquid chromatography (HPLC)/RIA analyses, and immunohistochemistry. Four antisera were employed: anti-SP that recognizes the amidated COOH-terminal of SP and is specific for the mature peptide, anti-SP4-10 that recognizes the midportion 4-10 amino acid sequence of SP and is highly specific for both mature SP and extended precursor forms of SP, anti-SP-G that is highly specific for the unamidated COOH-terminal of SP-G, and affinity-purified anti-SP-G-K that is capable of detecting SP-G and minor forms of SP precursor in immunohistochemical analyses. In all examined areas, the content of substance P4-10-like immunoreactivity (SP4-10-LI) quantified by RIA with the use of anti-SP4-10 was greater than that quantified by RIA with the use of anti-SP serum, thereby providing biochemical evidence of steady-state expression of extended precursor forms of SP. Immunohistochemical analyses demonstrated labeled axonal profiles indicating the presence of immunoreactive SP as well as immunoreactive forms of SP precursor within lung hilum and in small fibers in the parenchyma, with no evidence of labeled neuronal cell bodies in these same areas.

Amino Acid Sequence↗
PubMed1993

Distribution of tachykinin- and opioid-expressing neurons in the hamster solitary nucleus: an immuno- and in situ hybridization histochemical study.

In several sensory systems, tachykinin- and opioid-expressing neurons functionally interact and influence the processing of afferent information. To determine whether a similar relationship exists for the processing of general and special (gustatory) visceral afferent information, the present study mapped the distributions of these two neuronal phenotypes within the nucleus of the solitary tract (NST) of the hamster by employing a combination of immuno- and in situ hybridization histochemistry (ISHH). The hamster was chosen because it is frequently used as a model in taste studies, yet there is a relative dearth of data about peptide expression or the classical neurotransmitters in the brainstem of this animal. The immunohistochemical analyses employed 2 highly selective antisera directed towards the prototypical tachykinin and opioid peptides, i.e. substance P (SP) and methionine enkephalin (ENK), respectively. Intense staining of fibers and preterminal/terminal puncta was concentrated in the rostral pole or gustatory zone of the NST. SP-, but not ENK-like immunoreactivity was also observed in long courses of axon bundles traversing the brainstem enroute to the NST. Local application of colchicine engendered the appearance of a moderate number of SP-positive somata that were mostly clustered in the medial, central and intermediate subnuclei, as well as being scattered throughout the remainder of the NST, including the gustatory zone. A low number of isolated ENK-positive somata were also observed throughout the NST. The somal areas of the SP- and ENK-positive somata averaged 86.3 and 81.8 microns 2, respectively. The ISHH studies were performed using 2 selective oligodeoxynucleotide probes with complementary sequences to mRNAs encoding gamma-preprotachykinin (PPT) and preproenkephalin (PPE) molecules. Overall, the cellular expression of PPT mRNA within the NST corresponded both in distribution and in number to those identified by immunohistochemical analyses using anti-SP serum. In contrast, ISHH analyses monitored a significantly greater number of PPE-expressing somata in the medial, central, intermediate and ventrolateral nuclei than were ENK immunoreactive. These findings indicate that tachykinin and opioid peptide phenotypes are represented in neurons throughout the hamster NST and suggest a functional role for PPT- and PPE-related peptide forms in the modulation of afferent general visceral and gustatory information.

Animals↗
PubMed1993

Substance P markedly potentiates the antinociceptive effects of morphine sulfate administered at the spinal level.

The undecapeptide substance P and the alkaloid morphine sulfate are two agents previously thought to have opposite roles in the mediation of spinal nociceptive processes. The present report, however, demonstrates that low doses of substance P when coadministered with marginally effective doses of morphine sulfate into the rat subarachnoid space produce a markedly enhanced analgesic response, as monitored by the tail-flick test. This pharmacological effect is blocked by prior treatment with the opioid antagonist naloxone, indicating that the potentiated analgesic response is mediated by opioid-responsive neurons. In addition, the putative immediate precursor form of substance P (i.e., substance P-glycine) may substitute for the mature compound in the potentiated pharmacological effect. Moreover, the described synergism is unaffected by transection of the spinal cord, demonstrating the lack of supraspinal modulation of the observed phenomenon. Based on these observations, we are now able to dissociate opioid-potentiating and analgesic properties of substance P from traditional hyperalgesic effects realized at significantly higher concentrations. Consistent with previous biochemical data, a likely mechanism underlying the peptide-mediated enhancement of opioid analgesia may center on the ability of substance P to release endogenous opioid peptides within the local spinal cord environment. Finally, the pharmacological relationship of coadministered substance P and morphine sulfate established here supports the hypothesis that spinal tachykinin and opioid systems have a direct functional interaction in the modulation of local nociceptive responses.

Analgesia↗
PubMed1993

Detection and estimation of mRNA levels using a nonlinear model in neurons labeled by in situ hybridization histochemistry.

In situ hybridization histochemistry (ISHH) is an anatomical technique used to monitor gene expression at the cellular level via detection of steady-state levels of mRNA. Previously, densitometric analysis of ISHH-generated autoradiographic material has provided a relatively quantitative measure of the level of a specific mRNA distributed in any given anatomical region. The present study details the development of a parametric modeling technique used to automate the quantitative aspects of ISHH. The ISHH experiments described here utilized a specific DNA probe complementary to mRNA molecules encoding the neuropeptide substance P and related tachykinin peptides. A nonlinear model was used to describe the dark-field intensity pattern of labeled neurons. The model's parameters were then employed in detecting individually hybridized neurons and in estimating levels of preprotachykinin mRNA and associated cellular areas. Total mRNA content was quantified by relating the intensities described by the models to those obtained from 14C autoradiographic standards. Finally, the algorithm's performance was evaluated by comparing these estimates to those obtained from manual grain counts of labeled neurons. Overall, the parametric model presented here facilitates the process of performing quantitative analysis of hybridized neurons based on predetermined and unbiased morphological criteria.

Animals↗
PubMed1993

Selective in situ hybridization histochemical analyses of alternatively spliced mRNAs encoding beta- and gamma-preprotachykinins in rat central nervous system.

The present study describes the development of an in situ hybridization histochemistry (ISHH) procedure which was employed to selectively monitor cellular distributions of the 2 major alternatively spliced beta- and gamma-species of mRNA encoding preprotachykinin (PPT) molecules found in rat CNS. For these purposes, 2 custom-designed oligodeoxynucleotide probes were synthesized corresponding to complementary sequences of beta- and gamma-PPT mRNAs. In particular, the gamma-selective probe was demonstrated to hybridize to the contiguous regions of RNA flanking the splice site formed by exclusion of exon 4. Initially, Northern blot analyses performed in conjunction with appropriate specificity controls demonstrated selective hybridization of the 32P-labeled beta- and gamma-selective probes to single bands of approximately 1.2-1.3 kilobases in size, consistent with previously established values for rat brain beta- and gamma-PPT mRNAs. In anatomical studies, results obtained from absorptions using competing nonradiolabeled oligonucleotides defined the specificity and selectivity of both probes for targeting their respective species of mRNA immobilized within sections of brain tissue. Extensive ISHH analyses using both beta- and gamma-selective probes demonstrated similar patterns of cellular labeling in all of the examined CNS areas. In addition, data obtained from analyses of adjacent thin sections of the dorsal root ganglia (DRG) indicated that beta- and gamma-PPT mRNAs were colocalized within individual DRG neurons, thereby suggesting generalized coexpression at the cellular level of both forms of mRNA. These data were complemented by semi-quantitative analyses which yielded cellular or intrinsic molar ratios of beta- to gamma-PPT mRNA of approximately 1:2-1:3, consistent with those values previously determined by nuclease protection analyses. In sum, a reasonable hypothesis evolving from the anatomical studies in combination with previous biochemical data supports the existence of a strong homeostatic mechanism involved in the maintenance of relatively constant intrinsic molar ratios of beta- to gamma-PPT mRNA by tachykinin-expressing neurons. The biological relevance of this putative fundamental relationship is discussed in the context of posttranslational processing of PPT molecules and of expression of mature tachykinins.

Animals↗
PubMed1992

Development of an antiserum to the midportion of substance P: applications for biochemical and anatomical studies of substance P-related peptide species in CNS tissues.

This report describes the generation and biochemical characterization of a high-affinity antiserum that recognizes an epitope contained in the midportion sequence of substance P, i.e., substance P4-10. Designated A47, this reagent bound a variety of related peptide species containing the substance P4-10 sequence with apparent equipotency. A double radioimmunoassay procedure was developed that utilized A47, in combination with a traditional high-affinity COOH-terminally directed anti-substance P serum, to provide quantification of mature and immature forms of substance P in CNS tissues. Across most rat CNS areas, levels of substance P-like immunoreactivity were consistently 15% higher when monitored by analyses using A47 versus anti-substance P serum. In the dorsal root ganglia, an apparent enhancement in levels of substance P-like immunoreactivity of approximately 40%, when quantified by analyses using A47 versus anti-substance P serum, was observed; this most likely reflected the presence of an active biosynthetic pool of intermediate processing forms of substance P in this tissue. Coordinated HPLC/radioimmunoassay analyses of extracted dorsal root ganglia tissues demonstrated multiple peaks of immunoreactivity corresponding to mature substance P and to several of its precursor forms found in the normal biosynthetic pathway. Of the total recovered HPLC-fractionated immunoreactivities, that corresponding to the putative immediate precursor to substance P, i.e., substance P-glycine, was the predominant peak. In an additional series of HPLC/radioimmunoassay analyses, selective decreases in immunoreactive peaks corresponding to precursor forms of substance P were observed in dorsal root ganglia tissues from rats treated with the neurotoxic agent capsaicin. These results indicated decreased turnover of substance P as a consequence of drug treatment. Finally, initial immunohistochemical analyses employing affinity-purified A47 produced an unusual pattern of labeling characterized by well defined punctate terminal elements within the superficial aspects of the dorsal horn of the spinal cord.

Animals↗
PubMed1991

Biochemical characterization and anatomical distribution of a major form of unamidated precursor of substance P in rat brain.

Previous work from this laboratory has provided biochemical characterization of several posttranslational processing intermediates of the neuropeptide substance P (SP) in central nervous system (CNS) tissues, including the COOH-terminal glycine-extended dodecapeptide Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met-Gly (SP-G). SP-G is a major species of unprocessed SP found in rodent CNS tissues, and is the likely immediate precursor form of SP in the biosynthetic scheme. Here we present extensive characterization of the normal regional distribution of SP-G, as compared to SP, throughout the rat CNS via coordinated biochemical and morphological analyses. By radioimmunoassay (RIA), an approximate 10-fold variation in regional levels of SP-G-like immunoreactivity (SP-G-LI) was observed, ranging from 0.30 pmol/g in the amygdala, to 6.49 pmol/g in the medulla. On a normalized basis, the regional variation of unamidated precursor relative to mature peptide (SP-G-LI/SP-LI molar ratio) ranged from 0.30% in the amygdala to 5.15% in the dorsal root ganglia (DRG). Overall, the highest SP-G-LI/SP-LI ratios were found in DRG, medulla, and spinal cord, i.e. CNS areas associated with primary sensory afferent innervation via capsaicin-sensitive unmyelinated small diameter fibers. In addition, chromatographic and RIA analyses of extracted brain tissues indicated that the quantified immunoreactivities corresponding to SP, SP-G, as well as an additional COOH-terminal Gly-Lys-extended precursor, i.e., SP-G-K, displayed very similar chromatographic behavior as demonstrated for chemically authentic standards. These biochemical data were complemented by immunohistochemical analyses demonstrating a pattern of immunohistochemical staining for the presence of SP-G-LI as a defined subset of SP-LI-containing neural elements. Here, reaction product was localized to dendritic, axonal, and terminal neuronal elements in representative CNS regions of the rat, with relatively high levels of SP-G-LI found within anatomical areas containing a high density of sensory terminal structures. In an attempt to provide correlative functional anatomy, a group of rats was treated with colchicine, in order to differentially localize SP-LI- and SP-G-LI-containing somata after inhibition of axoplasmic transport. Most prominently, colchicine administration engendered immunohistochemical visualization of both SP-LI- and SP-G-LI-positive cells in mesencephalic and brainstem regions associated with stress, pain responses, and central control of autonomic function. Within this context, the coordinate expression of both SP-LI- and of SP-G-LI-positive somata in discrete brain areas is probably indicative of high ongoing rates of tachykinin synthesis coupled to utilization.

Amino Acid Sequence↗
PubMed1991

Substance P immunoreactivity in the superficial laminae of the hamster olfactory bulb.

The direct application of colchicine to the hamster olfactory bulb has engendered the appearance of a broader distribution of substance P-like immunoreactivity (SP-LI) than has been previously reported. In the glomerular layer, both external tufted and periglomerular cells were strongly immunopositive. These two classes could be identified by somal size and the presence and branching pattern of intraglomerular dendrites. SP-LI neurons were also identified in the external plexiform layer and many were similar to middle tufted cells. These findings demonstrate that substance P expression is not uniquely associated with classes of projection neurons, but is also found in periglomerular cells, a class of inhibitory local circuit neurons.

Animals↗
PubMed1990

Effect of early exposure to delta-9-tetrahydrocannabinol on the levels of opioid peptides, gonadotropin-releasing hormone and substance P in the adult male rat brain.

The effects of neonatal exposure to delta-9-tetrahydrocannabinol (THC) on the adult animal brain neurochemistry and pain perception were evaluated. Newborn rat pups were culled to a litter size of 8 (males and females) and treated either with THC (2 mg/kg) or oil (control) daily, during days 1-4 after birth. After weaning, the THC-treated males were housed 4 per cage. During the juvenile period (day 50), the THC-treated animals exhibited significantly lower baseline tail-flick values (a measure of pain perception) than the control. However, as adults, the THC-treated animals exhibited significantly higher sensitivity to pain following 5 mg/kg morphine challenge. Furthermore, the THC-treated animals had significantly elevated beta-endorphin and methionine-enkephalin levels in almost all the brain areas sampled for the study. In addition, the neonatally THC-treated rats exhibited significantly higher levels of substance P (SP) and significantly lower levels of gonadotropin releasing hormone (GnRH) in the anterior hypothalamus-preoptic area. The SP and GnRH levels did not differ among the THC-treated and control animals in the medial basal hypothalamus. The results of this study indicate that even a very low dose of THC administered during the neonatal period has a long-lasting effect on the brain neurochemistry. In particular, neonatal administration of THC appears to alter functioning of the endogenous opioid system.

Animals↗
PubMed1990

Disulfiram administration affects substance P-like immunoreactive and monoaminergic neural systems in rodent brain.

The biosynthetic enzyme peptidylglycine alpha-amidating monooxygenase catalyzes the formation of a variety of biologically active alpha-amidated peptides from respective COOH-terminal glycine-extended peptide precursors. Peptidylglycine alpha-amidating monooxygenase activity is dependent on copper, ascorbate, and molecular oxygen and is inhibited by the relatively selective copper chelator N,N-diethyldithiocarbamate or its disulfide dimer disulfiram (Antabuse). In the present study, chronic disulfiram treatment (100 mg/kg/day, for 12-25 days) resulted in significant changes in several neurochemical parameters in the mouse central nervous system, including levels of substance P-like, unamidated substance P-Gly-like, and protease-generated substance P-Gly-Lys-like immunoreactivities (SP-LI, SP-G-LI, and SP-G-K-LI, respectively). Combined high performance liquid chromatography/radioimmunoassay analyses of the extracted SP-LI, SP-G-LI, and SP-G-K-LI species indicated very similar chromatographic and immunochemical behavior as demonstrated for chemically authentic peptide standards. Additionally, changes in levels of monoamines and their metabolites were observed after drug administration. Complementary immunohistochemical analyses using affinity-purified anti-SP-G sera localized these drug-induced changes in levels of immunoreactive unamidated precursor to neural elements that normally express SP. As a functional corollary to alterations in neurochemical parameters, we observed significant disulfiram-induced increases in pain thresholds, potentiated by capsaicin treatment. Overall, our results indicate that the observed changes in steady state levels of immunoreactive SP and of the immature COOH-terminal extended forms of SP may reflect compensatory biosynthetic and posttranslational processing events in SP-containing neural systems after pharmacological challenge.

Amino Acid Sequence↗
PubMed1989

Spinal dynorphin involvement in the analgesia of pregnancy: effects of intrathecal dynorphin antisera.

In both rats and humans there is an analgesia associated with pregnancy. This analgesia is spinally mediated and involves the kappa type of opiate receptor. The current study demonstrates that intrathecal administration of high affinity dynorphin antibodies produces a significant reduction in jump thresholds during pregnancy (day 20). The administration of pre-adsorbed antisera fails to produce this effect. These results support the hypothesis that a spinal dynorphin/kappa opiate receptor system is activated during gestation.

Analgesia↗
PubMed1989

Sufentanil, morphine, met-enkephalin, and kappa-agonist (U-50,488H) inhibit substance P release from primary sensory neurons: a model for presynaptic spinal opioid actions.

An in vitro model system for analysis of presynaptic inhibitory actions of spinal opioids has been applied. Embryonic sensory neurons derived from chick dorsal root ganglia were grown in primary cell culture, and the release of substance P was evoked by electrical field stimulation during exposure to drugs with well-demonstrated affinity for opioid receptors. This allowed a pharmacologic characterization of the inhibitory actions of specific opioid agonists on the release of substance P as measured by radioimmunoassay (RIA). Sufentanil (0.5 microM), a high affinity mu receptor agonist, U-50,488H (25 microM), a selective kappa receptor agonist, and morphine (10 microM), an agonist with high affinity for mu and delta receptors, inhibited the evoked release of substance P by approximately 60%, 40%, and 50%, respectively. For sufentanil the response was demonstrated to be dose-dependent. As is the case for its analgesic action in vivo, morphine was approximately 50-fold less potent than sufentanil on a molar basis in this assay. The actions of sufentanil, U-50-488H and morphine were mimicked by the endogenous opioid peptide met-enkephalin, and its stable synthetic analog D-ala2-met5-enkephalinamide (DAME). Naloxone (25 microM), an opioid receptor antagonist, blocked the inhibitory action of sufentanil (0.5 microM), morphine (5 microM), and DAME (5 microM), but not U-50,488H (10 microM). The action of U-50,488H was partially blocked by the antagonist naltrexone (25 microM). Stereo-selectivity of agonist action was confirmed by the failure of dextrorphan (50 microM), an inactive opioid isomer, to inhibit the release of substance P.(ABSTRACT TRUNCATED AT 250 WORDS)

3,4-Dichloro-N-methyl-N-(2-(1-pyrrolidinyl)-cycloh↗
PubMed1989

G proteins couple alpha-adrenergic and GABAb receptors to inhibition of peptide secretion from peripheral sensory neurons.

Regulation of neuronal calcium channels by GTP-binding proteins (G proteins) is likely to be an important mechanism by which inhibitory transmitters influence excitation-secretion coupling in presynaptic nerve endings. Here, we report that in peripheral sensory neurons from embryonic chick dorsal root ganglia (DRG), the G protein-mediated inhibition of voltage-dependent calcium channels may best explain how norepinephrine (NE) and GABA inhibit the electrically evoked, calcium-dependent release of substance P (SP). As is the case for the previously reported inhibitory actions of these transmitters on DRG cell calcium channels, we demonstrate that NE and GABA inhibit peptide secretion through activation of alpha-adrenergic and GABAb receptors that are functionally coupled to pertussis toxin (PTX)-sensitive G proteins. Pretreatment of DRG cell cultures with PTX blocked the ability of NE and GABA to inhibit the release of SP, an action correlated with PTX-catalyzed ADP-ribosylation of membrane proteins with apparent molecular weight (Mr) of 40-41 kDa. Western immunoblot analysis of chick DRG cell membrane proteins using antisera directed against synthetic peptides corresponding to amino acid sequences predicted from cDNAs for PTX-sensitive G protein alpha subunits revealed a minimum of 2 Gi-like proteins (Mr 40 and 41 kDa) and a third Go-like protein (Mr 40 kD). Significantly, these findings implicate Gi- and/or Go-like GTP-binding proteins as mediators of presynaptic inhibition in peripheral sensory neurons.

Adenosine Diphosphate Ribose↗
PubMed1988

The mucosa mediates tachyphylaxis to leukotrienes C4, D4 and E4 in guinea pig trachea.

Effects of mucosal abrasion on tracheal smooth muscle leukotriene C4, D4 and E4 tachyphylaxis and on histamine contractions before and after four leukotriene exposures were studied. In intact tracheae, leukotriene C4 and E4 second exposure contractions were greater than first. Subsequent contractions showed tachyphylaxis. Leukotriene D4 contractions exhibited progressive tachyphylaxis. Mucosal abrasion potentiated initial leukotriene C4 and D contractions and eliminated leukotriene C4, D4 and E4 tachyphylaxis. Four leukotriene (10(-8) M) exposures reduced histamine (10(-4) M) responses in intact but not abraded preparations. Thus leukotriene C4, D4 and E4 release non-specific inhibitor(s) from tracheal mucosa.

Animals↗
PubMed1988

Characterization of the electrically evoked release of substance P from dorsal root ganglion neurons: methods and dihydropyridine sensitivity.

The mechanism by which dihydropyridines (DHPs) modulate the electrically evoked or KCI-induced release of substance P (SP) from embryonic chick dorsal root ganglion (DRG) neurons was investigated in the present study. The release of SP, as measured by radioimmunoassay (RIA), was characterized in terms of its dependence on extracellular calcium ion, its stimulus-response relationship, its sensitivity to the calcium-channel blocker omega conus toxin (omega-CgTx), and its modulation by the DHPs Bay K 8644 and nifedipine. Here it is reported that omega-CgTx (1 microM) blocked the electrically evoked release of SP. In contrast, the calcium-channel agonist Bay K 8644 (5 microM) facilitated the release of SP (by 45%), whereas the calcium-channel antagonist nifedipine (5 microM) was without effect. When the release of SP was triggered by depolarization of cultures with 60 mM KCI, the actions of the DHPs became much more pronounced. Under these conditions, Bay K 8644 facilitated (by 115%), whereas nifedipine inhibited (by 58%), peptide secretion. Voltage-clamp analysis of DRG cell calcium currents demonstrated that these actions of omega-CgTx, Bay K 8644, and nifedipine are explicable in terms of their effects on the slowly inactivating (L-type) calcium current. On the basis of these findings, it is suggested that the SP release mechanism exhibits DHP sensitivity due to the involvement of L-type calcium channels in the neurosecretory process. This model predicts that the voltage and time-dependent antagonist actions of nifedipine are sufficient to explain its failure to inhibit the electrically evoked release of SP.

3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗
PubMed1987

Transcriptional and translational requirements for developmental alterations in acetylcholine receptor channel function in Xenopus myotomal muscle.

Two functionally distinct types of acetylcholine (ACh) receptor channels are present on embryonic Xenopus myotomal muscle. During differentiation of this muscle, both in vivo and in dissociated cell culture, the occurrence of the high conductance "fast" channel type increases relative to the low conductance "slow" channel type. In order to ascertain whether new receptor synthesis is required for this rapid switch in channel types we examined the effects of inhibitors of transcription (alpha-amanitin and actinomycin D) and translation (cycloheximide) on developing muscle in culture. Inhibition of protein synthesis resulted in greater than 95% reduction in ACh receptor incorporation and also reversibly blocked the developmental appearance of the high conductance channel type. Inhibition of mRNA synthesis only slightly reduced the rate of receptor incorporation into muscle membrane over a 24-hr period but reversibly blocked appearance of the high conductance channel. These findings suggest that the high conductance ACh receptor channel type does not result from post-translational modifications of the low conductance type, but rather from transcription of a different mRNA encoding one or more of the ACh receptor subunits.

Amanitins↗