PC2 and 7B2 null mice demonstrate that PC2 is essential for normal pro-CCK processing.

Analysis of CCK content in extracts of whole forebrain from PC2 and 7B2 null mouse brain showed a significant decrease relative to wild-type brains. More detailed analysis revealed that CCK 8 amide levels in cerebral cortex and forebrain regions were more decreased than in hypothalamus. CCK 8 content in PC2 null mouse intestines was identical to control. Null mutant brains contained less CCK 8 than wild type and no other forms were seen when analyzed by gel filtration chromatography. No brain area examined was completely devoid of CCK, suggesting that other enzymes can partially compensate for the loss of PC2. This is the first demonstration that any endoprotease is important for CCK processing but also suggest the presence of a redundant system to ensure production of active CCK in the brain.

Pharmacological characterization of the release of neuropeptide Y-like immunoreactivity from the rat hypothalamus.

Radioimmunoassay for NPY and detection by HPLC-EC were used to detect the concurrent release of norepinephrine and NPY from slices of the rat hypothalamus and to examine the modulation by adrenoceptors of the release of this neuropeptide. Basal and potassium-evoked (56 mM K+) release of both compounds were easily measured, with evoked release occurring in a calcium-dependent manner. The effect of the alpha 2-adrenoceptor agonist clonidine, the antagonists prazosin (alpha 1 selective) and yohimbine (alpha 2 selective) and the beta-adrenoceptor antagonist propranolol were all shown to modulate the evoked release of NPY. The alpha 2 agonist clonidine decreased evoked release of NPY, while the alpha 2 antagonist yohimbine increased the potassium-evoked release. Prazosin decreased both the basal and potassium-evoked release of NPY. Propranolol had the most profound effect on release of NPY, causing a significant decrease in basal release and a large decrease in the potassium-evoked release of NPY from slices of hypothalamus.

Blood pressure increases after injection of neuropeptide Y into posterior hypothalamic nucleus.

Unilateral microinjection of neuropeptide Y (NPY; 0.235-2.35 nmol) into the posterior hypothalamic nucleus was found to evoke a concentration-dependent increase in mean arterial pressure (MAP) of Urethane-anesthetized rats. Concentration-dependent pressor responses were also elicited by unilateral administration of histamine (0.543-17.9 nmol) into the posterior hypothalamic nucleus. Administration of 30 nmol of the histamine H1-receptor antagonist, chlorpheniramine, but not 43.5 nmol of the histamine H2-receptor antagonist, cimetidine, into the posterior hypothalamic nucleus 10 min before 5.43 nmol histamine administration, significantly attenuated the histamine-induced pressor response. These concentrations of chlorpheniramine or cimetidine did not affect the increase in MAP, which could be evoked by the administration of 5.48 nmol of the cholinergic muscarinic agonist carbachol into the posterior hypothalamic nucleus. The carbachol-induced increase in MAP was, however, completely blocked by administration of 12 nmol of the cholinergic muscarinic antagonist atropine into the posterior hypothalamic nucleus 10 min before carbachol administration. This concentration of atropine did not affect the histamine-induced pressor response. Administration of atropine or chlorpheniramine into the posterior hypothalamic nucleus 10 min before 2.35 nmol NPY significantly attenuated the pressor response evoked by NPY. Cimetidine, on the other hand, was unable to significantly affect the increase in MAP evoked by NPY. These results demonstrate that NPY administered into the posterior hypothalamic nucleus can elicit a pressor response, and that this pressor response might involve local histaminergic and cholinergic neuronal pathways.

Cardiovascular effects and modulation of noradrenergic neurotransmission following central and peripheral administration of neuropeptide Y.

Experiments have been conducted to evaluate the effect of neuropeptide Y (NPY) administered at three distinct levels of the nervous system: 1) the posterior hypothalamic nucleus, 2) the spinal cord, and 3) the vascular noradrenergic neuroeffector junction. It was observed that NPY produced varying cardiovascular effects at these three distinct sites of the nervous system. Microinjections into the posterior hypothalamic nucleus resulted in an increase in blood pressure, which was reduced by prior microinjection of a muscarinic or H1-histamine antagonist but not an H2-histamine antagonist. In addition to the involvement of histaminergic and cholinergic pathways, the pressor effect of NPY appears to result from an increase in sympathetic outflow. NPY was also seen to decrease the potassium-induced release of norepinephrine (NE) from slices obtained from the posterior hypothalamic nucleus. In contrast to what was observed in the hypothalamus, the intrathecal injection of NPY at a level of T4 or T10 in anesthetized or T10 in unanesthetized rats resulted in a depressor effect as well as a decrease in heart rate. Both an alpha 2- and beta-adrenoceptor antagonist reduced the NPY effect. The depressor effect of intrathecal NPY was attenuated in rats pretreated with reserpine as well as in Spontaneously Hypertensive rats (SHR). These data suggest that the effects of NPY are closely associated with sympathetic preganglionic neurons in the spinal cord. At the vascular noradrenergic neuroeffector junction, NPY decreased the nerve stimulation-induced release of NE while potentiating the contractile response. Moreover, NPY potentiated the increase in perfusion pressure of the perfused mesenteric arterial bed in response to angiotensin, vasopressin, or phenylephrine.(ABSTRACT TRUNCATED AT 250 WORDS)

Increase in hypothalamic cholecystokinin following acute and chronic morphine.

Recent evidence supports an antagonistic interaction between cholecystokinin (CCK) and opiate peptides. The present study determined the effects of various levels of morphine treatment on hypothalamic levels of CCK as determined by radioimmunoassay. Acute treatment with morphine sulfate (10 mg/kg) or implantation of one morphine pellet (75 mg free base) increased levels of CCK in whole hypothalamus. Increased exposure to morphine by either chronic injections or implantation of two pellets did not result in a further change in whole hypothalamic CCK levels. In samples dissected into hypothalamic subregions, the effect of morphine on CCK levels was localized to medial but not lateral or posterior regions. These experiments extend earlier in vitro findings and suggest that some of the physiological and behavioral effects of opiate peptides may result from modulation of endogenous CCK.

The distribution of chromatographic characterization of an amino-terminal fragment of cholecystokinin (CCK) 58 in rat brain.

Utilizing a specific antiserum against CCK 58, a single immunoreactive peptide of about 1750 daltons was detected in rat brain extracts. It is distributed in all rat brain regions containing CCK 8, though it is most abundant in areas where CCK terminals predominate (septum, striatum and olfactory tubercle/nucleus accumbens). Based on its molecular weight, it is probably the amino terminal portion of CCK 58 left when CCK 39 is cleaved, and it may represent an intermediate in the processing of pre-pro-CCK. The presence of this peptide in CCK terminal areas implies that the proteolytic cleavage of CCK 58 occurs late in the processing, possibly in synaptic vesicles. It is also possible that this peptide can be released along with CCK 8 and exert an influence on synaptic transmission.

An overview of the biochemistry and anatomy of cholecystokinin (CCK) peptides in the brain.

In the nine years that have passed since the discovery of CCK in the brain, we have learned much about its biochemistry and anatomy. The anatomy of CCK in the brain is complex and in some instances does not fit with classical anatomical concepts. It is apparently common for some brain structures to have multiple CCK innervation and for interneurons and possible projection neurons to be present in the same structures, which may also receive afferent CCK input. Much work needs to be done before we completely understand the biochemistry, anatomy and function of CCK in the brain.

The distribution of cholecystokinin immunoreactivity in the central nervous system of the rat as determined by radioimmunoassay.

The regional distribution of cholecystokinin (CCK) in the rat brain was determined utilizing a radioimmunoassay which detects both gastrin and CCK. CCK concentration is highest in the caudate nucleus (10-14 ng CCK 8 equivalents/mg protein), followed by the cerebral cortex. Within the cerebral cortex, CCK is highest in the cingulate, pyriform, and entorhinal areas. There are substantial CCK concentrations in all other brain regions except pons, medulla and cerebellum. CCK is widely distributed in the hypothalamus, where it is highest in the median eminence and ventromedial nucleus. Considerable CCK-like immunoreactivity is also present in the posterior lobe of the pituitary gland, but is not detectable in anterior and intermediate lobes. Though the antisera used in this study cross-react with gastrin the dominant CCK-like material found in rat brain co-elutes with sulfated CCK 8 and separates from gastrin on Sephadex G-25 and HPLC chromatography.

Cholecystokinin in the central nervous system.

We have used high performance liquid chromatography and radioimmunoassay to characterize the CCK-like molecules in the central nervous system. The major form of CCK is the sulfated octapeptide. This molecule is distributed unevenly among various brain regions; the highest levels are found in the cortex and striatum. We have focused our attention on three areas in particular, the posterior pituitary, the caudate nucleus, and the hippocampus. The first of these receives its CCK-containing fibers from the hypothalamic magnocellular nuclei, the second appears to be innervated by the claustrum (or piriform cortex). The CCK in the hippocampus, on the other hand, is in intrinsic neurons. These structures and others in the brain have CCK binding sites.

Identification, characterization and distribution of motilin immunoreactivity in the rat central nervous system.

Motilin-immunoreactivity was evaluated in rat brain using 15 different antisera and by combining gel filtration, high pressure gel filtration and reverse phase high pressure liquid chromatography with radioimmunoassay. Gel filtration chromatography demonstrated a high molecular weight and low molecular weight form of immunoreactive motilin. The high molecular weight form predominated in brain while the low molecular weight peptide was the predominant form of duodenum. The low molecular weight immunoreactive motilin was indistinguishable from synthetic porcine motilin by gel filtration and high molecular weight gel filtration. Low molecular weight rat motilin could, however, be distinguished from synthetic porcine motilin by high pressure liquid chromatography and certain antisera. Immunological results suggest that the slight structural difference may be in the N-terminal portion of the molecule. Immunoreactivity was measured in grossly and microdissected regions of the rat brain. The peptide had quite a unique distribution as highest concentrations are observed in the cerebellum. High concentrations were also observed in hypothalamic nuclei. Particularly high concentrations were noted in the organum vasculosum lamina terminalis. Lowest motilin concentrations in the rat brain were in the pons and the medulla. The distribution of motilin in rat brain suggests that it may have roles in regulating both neuroendocrine and neurological processes.