Enkephalin-degrading dipeptidyl carboxypeptidase in guinea pig serum: its properties and action on bioactive peptides

A dipeptidyl carboxypeptidase, which cleaved the Gly3-Phe4 bond of enkephalins, was purified from guinea pig serum 420-fold. The optimum pH of the enzyme was in the neutral range (pH 7.25), and the molecular weight was estimated to be approx. 280,000. The enzyme hydrolyzed Met- and Leu-enkephalin with Km values of 0.30 and 0.50 mM, respectively. The enzyme was inhibited by metal chelators and p-chloro-mercuribenzoate. Captopril showed high inhibitory potency, while phosphoramidon and Phe-Ala showed no effect on the enzyme activity. Therefore, the obtained enzyme can be classified as an angiotensin-converting enzyme (EC 3.4.15.1). Among the bioactive peptides examined, bradykinin and angiotensin I were hydrolyzed by the enzyme. Angiotensin III showed a stronger inhibitory effect than that of angiotensin II. Substance P, gastrin I, and secretin were also inhibitory toward the enzyme activity. On high-performance liquid chromatography analysis, Met-enkephalin-Arg6-Phe7 and Leu-enkephalin-Arg6 were cleaved sequentially at the second peptide bond of the C terminus. Thus, the dipeptidyl carboxypeptidase in guinea pig serum may play a role not only in the angiotensin-bradykinin system but also in the metabolism of circulating enkephalins and other bioactive peptides.     

High-affinity 3H-substance P binding to longitudinal muscle membranes of the guinea pig small intestine

The binding of 3H-substance P (3H-SP) to longitudinal muscle membranes of the guinea pig small intestine has been characterized. The binding of 3H-SP exhibited a high affinity (Kd = 0.5nM). It was saturable (Bmax = 2 fmoles/mg tissue), reversible, and temperature-dependent. Kinetic studies and competition of 3H-SP binding by unlabeled SP yielded Kd and Ki values, respectively, which were in good agreement with the Kd calculated from saturation studies. The binding of 3H-SP appeared to be dependent on the presence of divalent cations in the incubation buffer. It was displaced by SP and various analogs and fragments in the rank order of SP greater than SP-(2-11) = SP-(3-11) greater than Nle11- SP = physalaemin greater than SP-(4-11) greater than SP-(5-11) greater than eledoisin much greater than SP-(7-11). Our results indicate that 3H-SP binds in longitudinal muscle of the guinea pig small intestine to a biologically relevant receptor which in many respects resembles the SP receptor characterized in the brain and the salivary gland of the rat.     

The longitudinal smooth muscle layer of the pig small intestine is innervated by both myenteric and submucous neurons

Originally, intestinal motility was thought to be exclusively regulated by myenteric neurons. Some years ago, however, it was demonstrated in large mammals that submucous neurons also participate in the innervation of the circular smooth muscle layer. To date, no information is available about the submucous innervation of the longitudinal smooth muscle layer (LM). This study provides evidence that in the small intestine of large mammals, the LM is innervated not only by the myenteric plexus, but also by the inner and outer submucous plexuses (ISP and OSP). In the porcine small intestine, the involved neurons can be subdivided into the following neurochemically distinct populations: leu-enkephalin (ENK)- and/or substance P (SP)-IR neurons and nitric oxide synthase (NOS)- and/or vasoactive intestinal polypeptide (VIP)-IR neurons. In the myenteric plexus, the majority of VIP- and/or NOS-IR neurons and ENK(+)/SP(-)-IR neurons exhibit descending projections, whereas ENK(+)/SP(+)-IR neurons preferentially have ascending projections. The ENK(-)/SP(+)-IR neurons do not show a polarized pattern. In the OSP, only ENK(+)/SP(-)- and VIP(+)/NOS(-)-IR neurons display a polarized (descending) projection pattern, whereas no polarization can be noted in the ISP. Morphological analysis of the traced neurons revealed that, in general, myenteric descending LM motor neurons have larger cell bodies than ascending ones and, in addition, myenteric descending VIP- and/or NOS-IR neurons have longer projections than ENK and/or SP-IR neurons. In conclusion, the present study demonstrates the involvement of not only myenteric, but also submucous neurons in the innervation of the LM. The two major populations are descending nitrergic neurons and ascending tachykinergic motor neurons, but also other subpopulations with specific projection patterns and neurochemical features have been identified.     

3d Mechanical properties of the partially obstructed guinea pig small intestine

Background and aimsPartial obstruction of the small intestine results in severe hypertrophy of smooth muscle cells, dilatation and functional denervation. Hypertrophy of the small intestine is associated with alteration of the wall structure and the mechanical properties. The aims of this study were to determine three dimensional material properties of the obstructed small intestine in guinea pigs and to obtain the 3D stress–strain distributions in the small intestinal wall.MethodsPartial obstruction of mid-jejunum was created surgically in five guinea pigs that were euthanized 2 weeks after the surgery. Ten-cm-long segments proximal to the obstruction site were used for the stretch-inflation mechanical test using a tri-axial test machine. The outer diameter, longitudinal force and the luminal pressure during the test were recorded simultaneously. An anisotropic exponential pseudo-strain energy density function was used as the constitutive equation to fit the experimental loading curve and for computation of the stress–strain distribution.ResultsThe wall thickness and the wall area increased significantly in the obstructed jejunum (P<0.001). The pressure—outer radius curves in the obstructed segments were translated to the left of the normal segments, indicating wall stiffening after the obstruction. The circumferential stress and the longitudinal stress through the wall were higher in the obstructed segments (P<0.02). This was independent of whether the zero-stress state or the no-load states were used as the reference state.ConclusionThe mechanical behaviour of the obstructed small intestine can be described using a 3D constitutive model. The obstruction-induced biomechanical properties change was characterized by higher circumferential and longitudinal stresses in the wall and altered material constants in the 3D constitutive model.     

Prostaglandin-norepinephrine interaction in guinea pig longitudinal muscle

Norepinephrine block of electrically induced contractions of the guinea pig longitudinal muscle-myenteric plexus preparation reverses spontaneously. PGE₁ or E₂ fails to alter rate of reversal in the presence of ascorbic acid but increases the rate in its absence. Using spectrophotometry, it could be demonstrated that PGE₁ or E₂ significantly increases the rate of autoxidation of norepinephrine, thereby accounting for the pharmacological interaction observed.     

Direct contractile effect of motilin on isolated smooth muscle cells of guinea pig small intestine.

We examined the direct effect of motilin on longitudinal and circular smooth muscle cells isolated from the guinea pig small intestine. In addition, the effects of 8-(N,N-diethylamino)-octyl-3,4,5-trimethoxy-benzoate hydrochloride (TMB-8, an inhibitor of intracellular Ca(2+)-release), verapamil (a voltage-dependent Ca(2+)-channel blocker), and removal of extracellular Ca2+ were investigated to evaluate the role of intracellular Ca2+ stores and extracellular Ca2+ on the muscle contraction induced by motilin. The effects of atropine (a muscarinic receptor antagonist), spantide (a substance P receptor antagonist) and loxiglumide (a CCK-receptor antagonist) were also examined to determine whether the motilin-induced contraction was independent of those receptors. Motilin induced a contraction of the longitudinal and circular smooth muscle cells in a dose-dependent manner with the maximal effect attained after 30 seconds of incubation. The ED50 values were 0.3 nM and 0.05 nM, respectively. TMB-8 suppressed completely the motilin-induced contraction of both types of smooth muscle cells. Verapamil had only a slight suppressive effect. Removal of extracellular Ca2+ did not have any significant influence on motilin-induced contraction. The contractile response to motilin was not affected by atropine, spantide or loxiglumide. Our findings showed that:1) motilin has a direct contractile effect on both longitudinal and circular smooth muscle cells; 2) this contractile effect is not evoked via muscarinic, substance P or CCK receptors, and 3) the intracellular release of Ca2+ plays an important role in the contractile response to motilin on both types of smooth muscle cells.     

Neuroimmune interactions in guinea pig stomach and small intestine

Enteric neuroimmune interactions in gastrointestinal hypersensitivity responses involve antigen detection by mast cells, mast cell degranulation, release of chemical mediators, and modulatory actions of the mediators on the enteric nervous system (ENS). Electrophysiological methods were used to investigate electrical and synaptic behavior of neurons in the stomach and small intestine during exposure to beta-lactoglobulin in guinea pigs sensitized to cow's milk. Application of beta-lactoglobulin to sensitized preparations depolarized the membrane potential and increased neuronal excitability in small intestinal neurons but not in gastric neurons. Effects on membrane potential and excitability in the small intestine were suppressed by the mast cell stabilizing drug ketotifen, the histamine H(2) receptor antagonist cimetidine, the cyclooxygenase inhibitor piroxicam, and the 5-lipoxygenase inhibitor caffeic acid. Unlike small intestinal ganglion cells, gastric myenteric neurons did not respond to histamine applied exogenously. Antigenic exposure suppressed noradrenergic inhibitory neurotransmission in the small intestinal submucosal plexus. The histamine H(3) receptor antagonist thioperamide and piroxicam, but not caffeic acid, prevented the allergic suppression of noradrenergic inhibitory neurotransmission. Antigenic stimulation of neuronal excitability and suppression of synaptic transmission occurred only in milk-sensitized animals. Results suggest that signaling between mast cells and the ENS underlies intestinal, but not gastric, anaphylactic responses associated with food allergies. Histamine, prostaglandins, and leukotrienes are paracrine signals in the communication pathway from mast cells to the small intestinal ENS.     

Electrical rhythmicity and spread of action potentials in longitudinal muscle of guinea pig distal colon

Using simultaneous intracellular recordings, we have characterized 1) electrical activity in the longitudinal muscle (LM) of isolated segments of guinea pig distal colon free to contract spontaneously and 2) extent of propagation of spontaneous action potentials around the circumference of the colon. In all animals, rhythmical spontaneous depolarizations (SDs) were recorded that are usually associated with the generation of action potentials. Recordings from pairs of LM cells, separated by 100 microm in the circumferential axis, revealed that each action potential was phase locked at the two electrodes (mean propagation velocity: 3 mm/s). However, at an increased electrode separation distance of 1 mm circumferentially, action potentials and SDs became increasingly uncoordinated at the two recording sites. No SDs or action potentials ever propagated from one circumferential edge to the other (i.e., 13 mm apart). When LM strips were separated from the myenteric plexus and circular muscle, rhythmically firing SDs and action potentials were still recorded. Atropine (1 microM) or tetrodotoxin (1 microM) either reduced the frequency of SDs or temporarily abolished activity, whereas nifedipine (1 microM) always abolished SDs and action potentials. Kit-positive interstitial cells of Cajal were present at the level of the myenteric plexus and circular and longitudinal muscle. In summary, SDs and action potentials in LM propagate over discrete localized zones, usually <1 mm around the circumference of the colon. Furthermore, in contrast to the classic slow wave, rhythmic depolarizations in LM appear to be generated by an intrinsic property of the smooth muscle itself and are critically dependent on opening of L-type Ca(2+) channels.     

Cholinergic neurons are involved in the effect of substance P on the circular muscle of the guinea pig small intestine.

The mode of action of the excitatory neuropeptide substance P was studied on the circular muscle of the guinea pig ileum in vitro. Atropine or tetrodotoxin strongly inhibited substance P-induced phasic contractions. The atropine-resistant part of the circular response was blocked by tetrodotoxin. A newly-developed method for quantitative evaluation revealed a rightward displacement of the substance P concentration-response curve, as well as a strong depression of the maximum effect, in the presence of atropine. These results indicate that cholinergic (and probably also non-cholinergic) excitatory neurons mediate phasic contractions due to substance P. The tonic component of the substance P-induced contraction was slightly reduced by atropine.     

Epithelial distribution of neural receptors in the guinea pig small intestine

Neural and paracrine agents, such as dopamine, epinephrine, and histamine, affect intestinal epithelial function, but it is unclear if these agents act on receptors directly at the enterocyte level. The cellular localization and villus-crypt distribution of adrenergic, dopamine, and histamine receptors within the intestinal epithelium is obscure and needs to be identified. Single cell populations of villus or crypt epithelial cells were isolated from the jejunum of adult guinea pigs. Enterocytes were separated from intraepithelial lymphocytes by flow cytometry and specific binding was determined using fluorescent probes. Alpha1-adrenergic receptors were located on villus and crypt intraepithelial lymphocytes and enterocytes. Beta-adrenergic receptors were found on villus and crypt enterocytes. Dopamine receptors were found on all cell types examined, whereas histamine receptors were not detected (<10% for each cell population). These studies demonstrated that (1) receptors for epinephrine and dopamine exist on epithelial cells of the guinea pig jejunum, (2) beta-adrenergic receptors are found primarily on villus and crypt enterocytes and (3) intraepithelial lymphocytes contain alpha1-adrenergic, but have few beta-adrenergic, receptors. The presence of neural receptors suggests that these agents are acting, at least in part, at the enterocyte or intraepithelial lymphocyte levels to modulate intestinal and immune function.     

Inhibitory action of atrial natriuretic factor on cholinergic and nonadrenergic, noncholinergic neurotransmission in guinea pig small intestine

We studied the effect of synthetic rat atrial natriuretic factor (ANF) (Ser 99-Tyr 126) on the isolated guinea pig proximal ileum. This preparation contained about one-third of the endogenous tissue ANF content which, for the most part, comes from the blood. ANF inhibited, in a dose-dependent manner, cholinergic twitch contractions (EC50 = 4.2 nM), nonadrenergic, noncholinergic (NANC) primary and rebound contractions and histamine-induced sustained tonic contraction (but not carbachol induced contraction) of the longitudinal muscle. Ascending enteric reflex (AER) contractions of the circular muscle were inhibited though not dose-dependently. We suggest pre- and post-synaptic actions of sustained intestinal tissue and blood ANF levels which may play a role in regulating motor activity and muscle tone of the small intestine.     

Slow wave and spike action potentials recorded in cell cultures from the muscularis externa of the guinea pig small intestine.

Intracellular recordings were obtained to investigate whether slow wave and spike type action potentials are present in cell cultures of the muscularis externa from the guinea pig small intestine. The muscularis externa of the small intestine was dissociated by using specific purified enzymes and gentle mechanical dissociation. Cells were plated on cover slips and maintained in culture for up to 4 weeks. Dissociated cells obtained in this way reorganized themselves in a few days to form small cell clumps showing spontaneous movements. Intracellular recordings of these clumps displayed both spike and slow wave type action potentials. Spikes were observed on top of some slow waves and were abolished by the addition of nifedipine or the removal of extracellular calcium. Slow waves, however, were nifedipine insensitive and temperature sensitive, and were abolished by octanol (a gap junction blocker) and forskolin (an adenyl cyclase activator). Slow waves were never observed in small clumps (<50 microm), suggesting that a critical mass of cells might be required for their generation. These observations demonstrated for the first time the presence of nifedipine-insensitive slow waves in cell cultures of the muscularis externa from the guinea pig small intestine. Cell cultures allow rigorous control of the immediate environment for the cells and this should facilitate future studies on the molecular and cellular mechanisms responsible for the slow waves in the gastrointestinal tract.     

ATP-induced currents in submucous plexus neurons of the guinea pig small intestine

ATP-induced membrane durrents in the submucous neurons of the guinea pig small intestine were studied using the whole-cell patch-clamp recording technique. Being applied at –50 mV. ATP activated an inward non-selective cationic current in 68.3% of the investigated neurons. An increase in ATP concentration within the 1–1,000 µM range resulted in the s-like increase in the amplitude of ATP-induced current. The EC₅₀ was 150.0±18.5 µM, while the Hill number was 1.6. The current was selectively activated by ATP and was not blocked by P2 purinoreceptor antagonist suramin (50–300 µM).,-Methylene-ATP (100–200 µM) and,-methylene-ATP (100–200µM), which are P2-purinoreceptor agonists, as well as adenosine (100–300 µM), exerted no effects. Reactive blue 2, if applied up to 4 min, enhanced ATP-induced current, while its longer application partially suppressed this current. In most submucous neurons, acetylcholine (ACh) likewise activated an inward cationic current. The amplitude of ACh-induced current was lower if ACh was applied during a long-lasting application of ATP than if ACh only was applied. Hexamethonium (50 µM), d-tubocurarine (20–40 µM), and trimethaphan (30 µM) completely and reversibly blocked ACh-induced currents, regardless of the presence of ATP, and did not affect ATP-induced currents. The results suggest that ATP-induced currents in submucous neurons are due to activation of a unique type of P2 purinoreceptors, which function in connection with nicotinic ACh receptors.Neirofiziologiya/Neurophysiology, Vol. 28, No. 2/3, pp. 100–110, March–June, 1996.     

Cholinesterases and choline acetyltransferase in the longitudinal muscle of the guinea pig ileum

Summary In order to gain insight into the possible role of the ACh-system in the smooth muscle cell, the presence of choline acetyltransferase, acetylcholinesterase and butyrylcholinesterase was studied in the longitudinal muscle of the guinea-pig ileum after the mechanical removal of Auerbach's plexus. Such treatment completely removes all nerve elements as confirmed by histochemistry and electron-microscopic examination.It was found that in the longitudinal muscle devoid of all nervous elements a substantial percentage of the activity of all three enzymes still remained. Ultrastructural localization of acetylcholinesterase and butyrylcholinesterase was observed on the sarcolemma, sarcoplasmic reticulum, nuclear membrane and invaginations of the sarcolemma. The localization of cholinesterases coincides with sites which are presumably involved in calcium movements during contraction and relaxation. It is well known that the depolarized smooth muscle responds to exogenous ACh with a reversible, calcium dependent contraction and it was suggested that ACh may act by increasing the influx of calcium through the cell membrane or by liberating calcium from its bound form.The presence of choline acetyltransferase and cholinesterase activities in the muscle cell proper, as well as the localization of cholinesterases on structures connected with calcium movements, support the coexistence of an intrinsic cholinergic mechanism in the smooth muscle.Part of this work was presented at the 6^th International Meeting of the International Society for Neurochemistry, Copenhagen, 1977