Stress-induced analgesia in μ-opioid receptor knockout mice reveals normal function of the δ-opioid receptor system

Stress-induced analgesia (SIA) was examined in wildtype and μ-opioid receptor knockout mice. We used thermal paw withdrawal (TPW) latency following a continuous 3-min swim in 20°C water, and found a significant increase in TPW latency in both wild-type and knockout mice. Pre-treatment prior to the swim with naltrindole, a selective δ-opioid receptor antagonist, blocked the increase in TPW latency in knockout mice. These results demonstrate an intact δ-receptor-mediated function of a physiologically-released endogenous agonist in the μ-opioid receptor knockout mouse. The present findings are in contrast with previous reports that analgesia induced by exogenous delta agonists is reduced in the knockout mice.     

Two novel related peptides, neuropeptide Y (NPY) and peptide YY (PYY) inhibit the contraction of the electrically stimulated mouse vas deferens

The action of two related peptides, neuropeptide Y (NPY) and peptide YY (PYY) on the mouse vas deferens was investigated. Both peptides potently inhibited in a dose dependent manner, muscle contractions evoked by electrical field stimulation of the intramural neurons but did not alter noradrenaline evoked contractions. They were also found to inhibit the potentiation of the twitch elicited by neurotensin and substance P. NPY and PYY were equipotent in the bioassay. This data suggests the possibility that NPY acts as a neuromodulator of noradrenaline release at a presynaptic level in the mouse vas deferens.     

Cholecystokinin releases β-endorphin from the anterior pituitary gland

The effect of cholecystokinin on the release of β-endorphin immunoreactivity from anterior pituitary quarters and from cultures of dispersed anterior pituitary quarters was investigated. Cholecystokinin (> 10⁻⁷ M) caused a slight release of β-endorphin immunoreactivity from the pituitary quarters only.It is concluded that the direct effect of cholecystokinin on the anterior pituitary is not likely to have a functional significance in the release of β-endorphin and the co-secreted ACTH.     

Comparison of G-protein activation in the brain by μ-, δ-, and κ-opioid receptor agonists in μ-opioid receptor knockout mice

Mice lacking the μ-opioid receptor gene have been developed by a gene knockout procedure. In this study, the activity of opioid receptor coupled G-proteins was examined to investigate whether there is a change in the extent of coupling for μ-, δ-, and κ-opioid receptors in μ-opioid receptor knockout mice. Selective agonists of μ- (DAMGO), δ- (DPDPE), and κ- (U-69,593) opioid receptors stimulated [³⁵S]GTPγS binding in the caudate putamen and cortex of wild-type mice. In contrast, only U-69,593 stimulated [³⁵S]GTPγS binding in these regions of μ-opioid receptor knockout mice. These results confirmed the absence of G-protein activation by a μ-opioid receptor agonist in μ-opioid receptor knockout mice, and demonstrated that coupling of the κ-opioid receptor to G-proteins is preserved in these mice. However, G-protein activation by the δ-opioid receptor agonist, DPDPE, was reduced in the μ-opioid receptor knockout mice, at least in the brain regions studied using autoradiography.     

Opposite effects of the δ- and μ-opioid receptor agonists on ventilation in conscious adult dogs

We studied ventilation and ventilatory pattern in adult unanesthetized dogs after intracisternal adminstration of morphiceptin analogue (MA) (Tyr-Pro-NMePhe-d-Pro-NH₂) and morphine sulfate (MS) which are μ-receptor opioid agonists and afterd-Ala-d-Leuenkephalin (DADLE), a preferential δ-receptor opioid agonist. DADLE induced a prolongation in expiratory time, T_e, and a reduction in instantaneous minute ventilation, V_t/T_tot, which lasted for about 2 h and was dose dependent. In contrast, MA and MS induced a striking decrease in T_e and tidal volume with a net increase in V_t/T_tot. Both MA and DADLE increased the number of sighs per unit time while morphine did not. Naloxone increased V_t/T_tot when used after both DADLE or MA but larger doses were required for an observable effect after DADLE than after MA. These data suggest that in the unanesthetized dog: (1) the effect of intracisternal opioids on ventilation and ventilatory pattern is not uniform and (2) the opioid μ-receptor subsystem may involve different neuronal pathways from those of the opioid δ-receptor subsystem to modulate breathing.     

Ultrastructural localization of δ-opioid receptor and Met-enkephalin immunoreactivity in rat insular cortex

The insular cortex has been implicated in the reinforcing properties of opiates as well as in the integration of responses to sensory-motor stimulation. Moreover, the δ-opioid receptor (DOR) and the endogenous opioid ligand, Met⁵-enkephalin (ENK) are known to be prominently distributed in insular limbic cortex. To examine the anatomical sites for opioid activation of DOR in rat insular cortex, we used immunoperoxidase for detection of an antiserum raised against a peptide sequence unique to the DOR alone, and in combination with immunogold-silver labeling for ENK. Light microscopy showed intense DOR-like immunoreactivity (DOR-LI) in pyramidal cells and interneurons in deep laminae, and in varicose processes in both superficial and deep layers of the insular cortex. Ultrastructural analysis of layers V and VI in insular cortex showed that the most prominent immunoperoxidase labeling for DOR was in dendrites. This labeling was associated with asymmetric excitatory-type junctions postsynaptic to unlabeled terminals. Dendritic DOR-LI was also distributed along selective portions of non-synaptic plasma membranes and subsurface organelles. In dually labeled sections, dendrites containing DOR-LI sometimes received synaptic input from ENK-labeled terminals or more infrequently colocalized with ENK. Other axon terminals were exclusively immunolabeled for DOR or more rarely contained both DOR and ENK immunoreactivity. Within labeled axon terminals, distinct segments of the plasma membrane and membranes of immediately adjacent synaptic vesicles showed the largest accumulation of the peroxidase reaction product for DOR. These results indicate that in rat insular cortex DOR is primarily heteroreceptive, but also serves an autoreceptive function on certain ENK-containing neurons. Our results also provide the first ultrastructural evidence that in rat insular cortex endogenous opioids interact through the DOR (1) to modulate the postsynaptic responses to other excitatory afferents and (2) to presynaptically regulate the release of other neurotransmitters. The modulatory actions on both ENK-containing and non-ENK-containing neurons may contribute significantly to the reinforcing properties of exogenous opiates acting on the DOR in limbic cortex.     

Characterization of mechanical withdrawal responses and effects of μ-, δ- and κ-opioid agonists in normal and μ-opioid receptor knockout mice

Clinical and experimental observations suggest that opiates can exert different influences on the perception of stimuli from distinct sensory modalities. Thermally-induced nociception is classically responsive to opiate agonists. μ-Opioid receptor-deficient transgenic mice are more sensitive to thermal nociceptive stimuli and morphine fails to attenuate the nociceptive responses to thermal stimuli in these animals. To enhance our understanding of opiate influences on mechanical sensitivity, we have examined withdrawal responses to a sequence of ascending forces of mechanical stimuli in mice with normal (wild type), half-normal (heterozygous) and absent (homozygous) μ-opioid receptor levels. We report data from mice examined without drug pretreatment or following pretreatment with morphine, the selective κ-opioid agonist, U50488H, and the selective δ-opioid agonist, DPDPE. Saline-pretreated mice of each genotype displayed similar, monotonically increasing frequency of withdrawal responses to the graded stimuli. Subcutaneously administered morphine produced a dose-dependent reduction in withdrawal responses in wild type and heterozygous mice, but had no significant effect in homozygous mice. Intraventricular administration of DPDPE also reduced the frequency of paw withdrawal (FPW) in wild type mice, but not in homozygous mice. In contrast, systemic U50488H produced a dose-dependent attenuation of paw withdrawal in both wild type and homozygous mice. These findings suggest that (1) interactions of endogenous peptides with μ-opioid receptors may not play a significant role in the response to mechanical stimuli in drug-free animals, and (2) deficiency of μ-opioid receptors has no functional consequence on the response to the prototypical κ-opioid receptor agonist, but decreases responses to the prototypical μ- and δ-opioid receptor agonists.     

Fentanyl, a μ-opioid receptor agonist, phase shifts the hamster circadian pacemaker

The phase-shifting effects of the μ-opioid receptor agonist fentanyl on the circadian timing system were investigated in the hamster. Fentanyl injections during the mid-subjective day induced phase advances of the hamsters’ wheel-running activity rhythm. The shifts were not accompanied by an increase in locomotor activity but instead a decrease of activity was often observed. A dose–response curve indicated that with increasing dosage, the response probability increased, while the magnitude of the induced shift remained stable. The present data suggest that there is some role for opioid regulation of the circadian system.     

Developmental, behavioral, and opiate receptor changes after prenatal or postnatal β-endorphin, CRF, or Tyr-MIF-1

Developmental and long-term behavioral effects of perinatal injection of beta-endorphin (BE), CRF and Tyr- Pro-Leu- Gly- NH₂ (Tyr-MIF-1) in male rats were investigated along with the possibility that opiate receptors may be altered by the injection of BE during this critical time. Daily injections of peptide were given to pregnant females (100 μg/rat) in the week before birth or to the offspring (50 μg/rat) of untreated mothers during the first week of life. Prenatal BE and CRF reduced body weight on day 1, in contrast to Tyr-MIF-1 which produced a significant increase over controls by day 7 as well as a slight but significant acceleration of eye opening. Among the postnatal treatments, CRF-treated animals showed the most dramatic changes. These included decreased body weight, accelerated eye opening, and, in adulthood, increased open field rearing behavior and a tendency for a monotonic body temperature response to low doses of morphine, in contrast to the biphasic response shown by controls. BE, when given to pregnant mothers, increased the number (B_max) of [³H]naloxone-labeled (mu) receptors in whole brains of offspring assayed on day 14, but it did not significantly alter [³H]d-Ala-d-Leu-enkephalin-labeled (delta) receptors. In contrast, a significant decrease in both mu and delta receptors was observed on day 14 in rats given BE postnatally. These differences in receptors were no longer apparent in adulthood, and no significant differences in tail-flick response were detectable at this time. Nevertheless, some of the effects of these three peptides endured well beyond their presence, and for BE included changes in the number of opiate receptors.     

Increased affinity of dimeric enkephalins is not dependent on receptor density

The equilibrium binding and dissociation kinetics of the enkephalin dimer bis-(D-Ala2-D-Leu5-enkephalin)-ethylenediamide (designated DPE2) to neuroblastoma glioma NG108-15 cells were investigated and compared with the monomers D-Ala2-D-Leu5-enkephalin (DADL) and D-Ala2-Leu5-enkephalinamide (DALEA). Binding was studied after exposure of the membrane to increasing concentrations of the irreversible delta receptor selective ligand FIT in order to decrease the density of binding sites on the cell membrane. The increased affinity of DPE2 did not revert to that of the monomer DADL by this reduction of binding sites. Similarly, the dissociation of DPE2 did not approach that of the monomer DALEA in the presence of 1 microM DALEA. These data strongly suggest that crosslinking does not occur, and fail to confirm the hypothesis that dimers with short spanning chain length aid the clustering of receptors. We postulate: 1) If the dimer binds to a bivalent binding site, the monovalent binding state of our bivalent ligand may not exist to an appreciable extent, and 2) the bivalent ligand cannot bind when the binding site is irreversibly blocked by a monovalent ligand.     

Streptozotocin-induced diabetes selectively alters the potency of analgesia produced by μ-opioid agonists, but not by δ- and κ-opioid agonists

To investigate the possible mechanisms of the alterations in morphine-induced analgesia observed in diabetic mice, we examined the influence of streptozotocin-induced (STZ-induced) diabetes on analgesia mediated by the different opioid receptors. The antinociceptive potency of morphine (10 mg/kg), administered s.c., as determined by both the tail-pinch and the tail-flick test, was significantly reduced in diabetic mice as compared to that in controls. Mice with STZ-induced diabetes had significantly decreased sensitivity to intracerebroventricularly (i.c.v.) administered μ-opioid agonists, such as morphine (10 μg) and [d-Ala², N-Me Phe⁴,Gly-ol⁵]enkephalin (DAMGO, 0.5 μg). However, i.c.v. administration of [d-Pen^2,5]enkephalin (DPDPE, 5 μg), a δ-opioid agonist, and U-50,488H (50 μg), a κ-opioid agonist, produced pronounced antinociception in both control and diabetic mice. Furthermore, there were no significant differences in antinociceptive potency between diabetic and control mice when morphine (1 μg), DAMGO (10 μg), DPDPE (0.5 μg) or U-50,488H (50 μg) was administered intrathecally. In conclusion, mice with STZ-induced diabetes are selectively hyporesponsive to supraspinal μ-opioid receptor-mediated antinociception, but they are normally responsive to activation of δ- and κ-opioid receptors.     

The δ-opioid signal transduction on the gonadotropin-releasing hormone release is eicosanoid dependent

In static incubations, the K⁺ induced release of gonadotropin-releasing hormone (GnRH) and of prostaglandin (PG) E₂, was 2–3 times higher in the isolated median eminence (ME) compared to the hypothalamic area containing the arcuate nucleus (ARN) plus the ME. The δ-opioid agonist DTLET, induced a parallel, dose-dependent reduction of GnRH and PGE₂ release in the ARN plus ME. Both effects of DTLET were blocked by the δ-opioid antagonist Diallyl-G. In the isolated ME, DTLET reduced the secretion of PGE₂ but enhanced the release of GnRH. In this area Diallyl-G had no effect on the PGE₂ release but blocked the GnRH secretion. When the PGE₂ production was blocked by indomethacin in the ARN plus ME preparation, DTLET increased the release of GnRH and induced the production of leukotrienes (LTs). On the other hand, DTLET decreased the release of both GnRH and PGE₂ in the presence of nordihydroguaiaretic acid (NDGA), an inhibitor of the production of LTs. The above results suggest that: (a) the δ-opioid agonist DTLET modulates GnRH release differentially in the hypothalamic areas examined; and (b) the arachidonic acid metabolites are involved in the mode of action of DTLET on the release of GnRH in the ARN plus ME hypothalamic fragment.     

Opioid receptor subtypes in the rat spinal cord: electrophysiological studies with μ- and δ-opioid receptor agonists in the control of nociception

We have compared the ability of selective mu- and delta-opiate agonists to modulate nociceptive transmission at the level of the rat dorsal horn using electrophysiological approaches. Single-unit extracellular recordings were made from neurones in the lumbar dorsal horn of the intact rat under halothane anaesthesia. Neurones could be activated by both A- and C-fibre electrical stimulation (and by natural innocuous and noxious stimuli). Agonists were applied directly onto the cord in a volume of 50 microliters. The intrathecal administration of 3 agonists, Tyr-D-Ala-Gly-MePhe-Gly-ol (DAGO) (mu-selective) (2 X 10(-3)-10 nmol) Tyr-D-Thr-Gly-Phe-Leu-Thr (DTLET) (mu/delta) (7 X 10(-4)-70 nmol), and cyclic Tyr-D-Pen-Gly-Phe-D-Pen (DPDPE) (delta) (2 X 10(-2)-100 nmol) produced dose-dependent inhibitions of C-fibre-evoked neuronal activity whilst A-fibre activity was relatively unchanged. DAGO produced near-maximal inhibitions which could be completely reversed by naloxone (1.5 nmol) whilst DPDPE causes less marked inhibitions which could only be partially reversed by naloxone (1.5-13.5 nmol). DTLET produced effects intermediate to those of DAGO and DPDPE. The results suggest that both mu- and delta-opioid receptors can modulate the transmission of nociceptive information in the rat spinal cord.     

μ- and δ-opioid receptor mRNAs are expressed in spinally projecting serotonergic and nonserotonergic neurons of the rostral ventromedial medulla

The rostral ventromedial medulla (RVM) is an important mediator of the supraspinal component of opioid antinociception. Previous studies have suggested that activation of the cloned μ- and δ-opioid receptors (MOR1 and DOR1 respectively) in the RVM produces the antinociception mediated by spinally projecting neurons. In the present study, we investigated the expression of mRNA encoding either MOR1 or DOR1 in the RVM of rats. In addition, we examined quantitatively the expression of MOR1 and DOR1 mRNAs in spinally projecting RVM neurons including serotonergic (5HT) cells by using in situ hybridization, immunocytochemistry, retrograde tract-tracing, and the physical disector. Brainstem neurons were labeled in 14 male Sprague-Dawley rats by applying Fluoro-Gold (FG) topically to the dorsal surface of the lumbosacral spinal cord. Five-micrometer-thick cryostat sections were cut and in situ hybridization was performed by using full-length cRNA probes labeled with ³⁵S-UTP. We found that 43% of RVM projection neurons expressed MOR1 mRNA and 83% of RVM projection neurons expressed DOR1 mRNA. Of 192 retrogradely labeled cells in the RVM, 51 cells (27%) were immunoreactive for 5HT. Of this population, half appeared to be labeled for the mRNA encoding MOR1 and over three-fourths appeared to be labeled for the mRNA encoding DOR1. Thus, we conclude that bulbospinal neurons express MOR1 and DOR1; moreover, MOR1 and DOR1 are expressed by significant proportions of 5HT neurons projecting to or through the dorsal spinal cord. J. Comp. Neurol. 404:183–196, 1999. © 1999 Wiley-Liss, Inc.     

Quantitative autoradiography of μ-,δ- and κ1 opioid receptors in κ-opioid receptor knockout mice

Mice deficient in the κ-opioid receptor (KOR) gene have recently been developed by the technique of homologous recombination and shown to lack behavioural responses to the selective κ₁-receptor agonist U-50,488H. We have carried out quantitative autoradiography of μ-, δ- and κ₁ receptors in the brains of wild-type (+/+), heterozygous (+/−) and homozygous (−/−) KOR knockout mice to determine if there is any compensatory expression of μ- and δ-receptor subtypes in mutant animals. Adjacent coronal sections were cut from the brains of +/+, +/− and −/− mice for the determination of binding of [³H]CI-977, [³H]DAMGO ( -Ala²-MePhe⁴-Gly-ol⁵ enkephalin) or [³H]DELT-I ( -Ala² deltorphin I) to κ₁-, μ- and δ-receptors, respectively. In +/− mice there was a decrease in [³H]CI-977 binding of approximately 50% whilst no κ₁-receptors could be detected in any brain region of homozygous animals confirming the successful disruption of the KOR gene. There were no major changes in the number or distribution of μ- or δ-receptors in any brain region of mutant mice. There were, however some non-cortical regions where a small up-regulation of δ-receptors was observed in contrast to an opposing down-regulation of δ-receptors evident in μ-knockout brains. This effect was most notable in the nucleus accumbens and the vertical limb of the diagonal band, and suggests there may be functional interactions between μ- and δ-receptors and κ₁- and δ-receptors in mouse brain.     

Molecular cloning and expression of a δ-opioid recetpor from rat brain

We have isolated and characterized a rat δ-opioid receptor. The deduced amino acid sequence (372 aa) closely resembles the murine δ-opioid receptor, DOR-1. In fact, 97% of the amino acid residues are conserved between the two species, while 93% of the nucleic acid residues are identical. A 6 kb mRNA was detected in rat cortex using rat DOR-1 as a probe. When expressed in COS cells, the clone shows high-affinity opioid binding with selectivity for δ-opioids. The rat δ-opioid receptor cDNA clone will be a useful tool for studying the function of δ-opioid receptor in rats. Wiley-Liss, Inc.     

Imaging of δ- and μ-opioid receptors in temporal lobe epilepsy by positron emission tomography

The involvement of opioid neurotransmitter systems in seizure mechanisms is well documented. In previous positron emission tomography (PET) studies in patients with unilateral temporal lobe epilepsy, we have found evidence for differential regulation of the opioid-receptor subtypes. The present study extends our previous observations to δ-opioid receptors by using the δ-receptor-selective antagonist [¹¹C]methylnatrindole ([¹¹C]MeNTI). Paired measurements of δ- and μ-opioid receptor binding and metabolic activity were performed with PET using [¹¹C]MeNTI and [¹¹C]carfentanil ([¹¹C]CFN) and [¹⁸F]fluorodeoxyglucose ([¹⁸F]FDG), respectively. Binding of [¹¹C]MeNTI and [¹¹C]CFN increased and [¹⁸F]FDG uptake decreased in the temporal cortex (TC) ipsilateral to the focus. Decreases in [¹⁸F]FDG uptake were more widespread regionally than were increases in opioid receptors. Increases in the δ- and μ-receptor binding showed different regional patterns. Increases in δ-receptor binding were confined to the middle aspect of the inferior TC, whereas binding of δ receptors increased in the mid-inferior TC and anterior aspect of the middle and superior TC. The increase in δ receptors suggests their anticonvulsant action, as previously shown for the δ-receptor subtype, whereas the different regional pattern of receptor alterations suggest the distinct roles of different opioid-receptor subtypes in seizure phenomena.     

Localization of μ- and δ-opioid receptors in cat respiratory areas: an autoradiographic study

Autoradiography after in vitro binding with selective ligands for either mu ([3H](Tyr-D-Ala-Gly-(NMePhe)-Gly-ol] or delta ([3H](Tyr-D-Thr-Gly-Phe-Leu-Thr)) opioid receptor types revealed the presence of variable amounts of radioactive labeling in the cat brainstem. Areas involved in the respiratory rhythmogenesis were among the most prominently labeled structures. The pneumotaxic center, including the nucleus parabrachialis medialis and the Kolliker-Füse nucleus, contains a very high density of delta binding sites while the dorsal respiratory nucleus which corresponds to the nucleus tractus solitarius, is more heavily labeled by the mu ligand. The neuroanatomical differences in the distribution of opioid receptors correlates well with the pharmacological responses induced by administration of specific mu- or delta ligands.     

Regional,developmental, and cell cycle-dependent differences in μ, δ, and κ-opioid receptor expression among cultured mouse astrocytes

The diversity of opioid receptor expression was examined in astrocytes in low-density and non-dividing (confluent) cultures from the cerebral cortex, hippocampus, cerebellum, and striatum of 1-day-old mice. μ, δ, and κ opioid receptor expression was assessed in individual cells immunocytochemically, by using flow cytometry, and functionally by examining agonist-induced changes in intracellular calcium ([Ca²⁺]_i). Significant spatial and temporal differences were evident in the pattern of expression of μ, δ, and κ receptors among astrocytes. In low-density cultures, greater proportions of astrocytes expressed μ-opioid receptor immunoreactivity in the cerebral cortex and hippocampus (26–34%) than in the cerebellum or striatum (7–12%). At confluence, a greater percentage of astrocytes in cerebellar (26%) and striatal (30%) cultures expressed μ-immunoreactivity. Fewer astrocytes possessed δ-immunoreactivity in low-density striatal cultures (8%) compared to other regions (16–22%). The proportion of δ receptor-expressing astrocytes declined in the cerebellum but increased in the hippocampus. κ-opioid receptors were uniformly expressed by 27–34% of astrocytes from all regions, except in cortical cultures, where the proportion of κ expressing cells was 38% at low-density and decreased to 22% at confluence. Selective μ (PLO 17; H-Tyr-Pro-Phe (N-Me) -D-Pro-NH₂, δ ([D-Pen², D-Pen⁵] enkephalin), or κ (U50,488H; trans-(±)-3,4-Dichloro-N-methyl-N-[2-(1-pyrrolidinyl) cyclohexyl] benzeneacetamide methanesulfonate) opioid receptor agonists increased [Ca²⁺]_i in subpopulations of astrocytes indicating the presence of functional receptors. Lastly, opioid receptor immunofluorescence varied during the cell division cycle. A greater proportion of astrocytes in the G₂/M phase of the cell cycle were μ or δ receptor immunofluorescent than at G₀/G₁. When astrocytes were reversibly arrested in G₁, significantly fewer cells expressed δ receptor immunofluorescence; however, upon reentry into the cell cycle immunofluorescent cells reappeared. In conclusion, opioid phenotype varies considerably among individual cultured astrocytes, and this diversity was determined by regional and developmental (age and cell cycle dependent) differences in the brain. These in vitro findings suggest astroglia contribute to regional and developmental idiosyncrasies in opioid function within the brain. GLIA 22:249–259, 1998. © 1998 Wiley-Liss, Inc.     

Mechanism of action of cholecystokinin on intestinal blood flow; interactions with opioid peptides and vasoactive intestinal peptide

The effects of local administration of CCK-OP on mesenteric blood flow was studied in anaesthetized dogs using an electromagnetic flowmeter and interactions with the opioid peptides and VIP examined. CCK-OP was found to enhance enteral blood flow and tissue metabolism. Investigations with pharmacological antagonists showed that the vasoregulatory effect was exerted neurogenically, by influencing the activities of sympathetic alpha 2 adrenergic, dopaminergic, cholinergic, muscarinergic and nicotinic neurones. The flow-increasing action of CCK-OP was enhanced by D-Met2-NleS5-enkephalinamide; the effect was additive and partially blocked by naloxone. D-Met2-Pro5-enkephalinamide blocked the increases caused by CCK-OP in blood flow and tissue acetylcholine levels. The blockade was not competitive and could be totally eliminated by naloxone. VIP blocked the flow-increasing effect of CCK-OP by a non-competitive mechanism. The results provide data on the peptidergic regulatory mechanisms of the mesenteric circulation.