Detlef Bieger

Acetylcholinesterase in pontomedullary catecholamine neurons of the adult albino rat

This study investigated the cholinesterasic reactivity of catecholamine neurons in the rat hindbrain with the aid of a two-step histochemical procedure. First, catecholamine cells were visualized by their formaldehyde/glutaraldehyde induced specific histofluorescence and then poststained in the same tissue with a thiocholine technique for acetylcholinesterase (AChE). Processing the vibratome-sectioned tissue in phosphate buffer subsequent to initial aldehyde fixation permitted satisfactory preservation of both amine fluorophores and esterasic reactivity. Our results, in both randomly sampled and serially sectioned material, unequivocally establish the presence of AChE in all pontomedullary cell groups emitting catecholamine fluorescence, the majority of which are known to consist of noradrenaline perikarya. Hence in contrast to previous reports the occurrence of AChE in central noradrenaline neurons appears to be generalized. The intensity of histofluorescence and esterasic staining were uncorrelated in most regions. It remains for future study to determine whether AChE in brain catecholamine neurons indicates their cholinoceptivity or subserves the catabolism of other neuromediators such as substance P.

Inhibition of field stimulation-evoked relaxations in rat oesophageal smooth muscle by the calcium antagonist PN 200–110

1 The inhibitory effects of the 1,4‐dihydropyridine calcium channel antagonist, PN 200–110 (isradipine), on field stimulation‐evoked tetrodotoxin (TTX)‐sensitive and‐insensitive relaxations were studied in rat oesophageal smooth muscle of the tunica muscularis mucosae. 2 The TTX‐insensitive relaxation was inhibited by PN 200–110 in a stereoselective manner with the (+)−(S)‐isomer displaying a 1000 fold greater inhibitory potency than the (—)−(R) isomer. A similar potency was noted for inhibition of high K+‐evoked contractions. 3 TTX‐sensitive relaxations evoked by field stimulation and contractions elicited by the muscarinic cholinoceptor agonist, cis‐2‐methyl‐4‐dimethylamino‐methyl‐1,3‐dioxolane methiodide (cis‐dioxolane) were considerably less sensitive to inhibition by PN 200–110, although, again, stereoselectivity for PN 200–110 was apparent. 4 Pretreatment with (+)−(S)‐PN 200–110 resulted in a non‐competitive displacement of the Ca2+ concentration‐response curves obtained in the presence of either isotonic 50 mm KCl or cis‐dioxolane. The effect of K+ was 10 fold more sensitive than that of cis‐dioxolane. 5 The potency rank orders for inhibition of TTX‐insensitive field stimulation‐evoked relaxations and K+‐mediated contractions in a series of calcium channel antagonists were closely correlated; (+)−(S)‐PN 200–110 showing highest potency followed by nifedipine, verapamil, diltiazem, (—)−(R)‐PN 200–110. 6 It is concluded that TTX‐insensitive relaxations are dependent upon an influx of extracellular Ca2+ through potential‐operated calcium channels.

Atypical indolamine-immunoreactive cell groups in the dorsal myelencephalon of the rat

The presence of atypical indolamine-immunoreactive (IAI) neurones in the dorsal myelencephalon of the rat was demonstrated by means of peroxidase-antiperoxidase (PAP) immunocytochemistry. Besides the area postrema, two other regions, viz. the solitary complex and the superficial rostral cuneate fascicle, were found to contain neuronal perikarya displaying a normally weak staining which was markedly enhanced after monoaminoxidase (MAO) inhibition. In contrast to immunoreactive cells of the periaqueductal central gray, the dorsal myelencephalic IAI neurons were undetectable after serotonin synthesis inhibition with p-chlorophenylalanine (PCPA), as were immunoreactive terminal neuropils in most brainstem areas. However, sequential treatment with the MAO inhibitor, iproniazid, completely reversed the PCPA-induced suppression of perikaryal immunoreactivity and partially restored axonal staining. None of the atypical cell groups displayed a detectable formaldehyde-induced specific histofluorescence. Since brain levels of tryptamine are likely to increase significantly after MAO inhibitor/PCPA treatment, and furthermore tryptamine can be assumed to cross-react with serotonin, it is suggested that the observed atypical IAI neurons may represent either a subpopulation of serotoninergic neurons; previously postulated true tryptamine neurons; or non-indolaminergic neurons endowed with a selective uptake mechanism for serotonin or tryptamine. These results corroborate the view that different types of indolamine neurons exist in the rat brainstem. Moreover, they underscore the need for cautious interpretation of serotonin neuron mapping studies involving the use of MAO inhibitors.

Agonist‐induced periodic vasomotion in rat isolated pulmonary artery

Vasomotion is linked to the rapid oscillations of intracellular calcium levels. In rat pulmonary artery, this activity can manifest as a slow periodic on–off pattern, the timing of which depends on the type and intensity of pharmacological stimuli employed. In this study, we have sought to characterize a slow‐wave vasomotor activity pattern induced in isolated arterial ring preparations by simultaneous exposure to the α1‐adrenoceptor agonist phenylephrine (1–10 nm) and the L channel agonist S(‐)‐Bay K 8644 (3–20 nm). Treated tissues responded with a stable on–off pattern of vasomotion persisting for >5 h at 5–6 cycles/h. In intact rings, this response was suppressed by methacholine and restored or enhanced by Nω‐nitro‐l‐arginine methyl ester. Analogous inhibitory effects were obtained with high Mg2+, 8‐Br‐cGMP (but not 8‐Br‐cAMP), riluzole, ryanodine, chelerythrine, and fasudil. Pinacidil (30 nm) increased off‐cycle length without change in slow‐wave amplitude. Conversely, tetraethylammonium (1.0–3.0 mm) augmented the latter without affecting periodicity. Carbenoxolone (10 μm) abolished slow‐wave activity, while raising basal tone and inducing random phasic activity. In endothelium‐denuded rings, the threshold of agonist‐induced slow‐wave vasomotion was lowered and a similar inhibitory effect obtained with carbenoxolone. In conclusion, the slow‐wave pattern of vasomotion described here is (i) subject to inhibitory modulation by endothelial NO and an array of voltage‐gated and leak K conductances yet to be fully characterized; (ii) dependent on Ca2+ from both extracellular and sarcoendoplasmatic sources; (iii) controlled by kinase (Rho and PKC)‐mediated regulation of myosin light chain phosphatase; and (iv) synchronized via intermyocyte gap junctions.

Effects of vagal cooling on esophageal cardiovascular reflex responses in the rat.

Distension of the distal esophagus in the anesthetized rat causes a vagally-mediated arterial pressor and tachycardia response. To investigate the nature of viscerosensory fibers in the afferent limb of this reflex, the present study was carried out in urethane-anesthetized rats that were subjected to graded cooling of both cervical vagal trunks in situ. Distal esophageal distension was applied for 20 s by means of a water-filled high compliance balloon. Vagal cooling to 9 degrees C abolished pressor responses and unmasked a depressor component during maximal distension. Cooling to 7.5 degrees C blocked this inhibitory component, well above the temperature known to block C-fibers. We conclude that the cardiovascular response to esophageal distension is triggered via at least two subpopulations of A(delta) type vagal afferents that project to brain stem nuclei regulating central vasomotor tone.

Photosensitization of oesophageal smooth muscle by 3-NO2- 1,4-dihydropyridines: evidence for two cyclic GMP-dependent effector pathways

1 Photoactivated mechanical responses that resulted from exposure to 3‐NO2‐1,4‐dihydropyridines (3‐NO2‐DHPs) or NO‐donors were examined in rat isolated oesophageal smooth muscle with a view to determining the role of calcium and cyclic GMP. 2 Isometric contractile force was recorded in preparations bathed in normal Tyrode or 110 mM K+‐depolarizing solution. Exposure to (+)‐PN 202 791, (±)‐Bay K 8644 and (−)−PN 2020 791 or the photodegradable NO‐donors, sodium nitroprusside (SNP), streptozotocin (STZ) and sodium nitrite photosensitized precontracted tunica muscularis mucosae preparations in a concentration‐dependent fashion. Photosensitizing potency followed the order: (+)‐PN 202 791 > (±)‐Bay K 8644>(−)−PN 202 791 > SNP > STZ > NaNO2. 3 A low amplitude, slow photorelaxation (slope: 1 mg s−1) was obtained with the L‐channel antagonists (−)−PN 202 791 and (+)‐Bay K 4407. Photosensitization by the agonist enantiomers (+)‐ PN 202 791 and (−)−Bay K 5407, as well as racemic Bay K 8644, was mimicked by NO donors and showed at least three different components, consisting of (i) a fast relaxation (slope: 140 mg s−1), (ii) a fast ‘off‐contraction’, and (iii) a delayed slow relaxation. The fast components, but not the delayed slow relaxation, were abolished by blockade of L‐type voltage‐operated calcium channels, chelation of extracellular calcium and skinning of the plasmalemma, suggesting their mediation by a process linked to calcium entry through L‐channels. 4 Both cyclopiazonic acid (3–30 μm) and ryanodine (30 μm) inhibited the fast reponse. This inhibition was accelerated in the presence of extracellular calcium and resembled that seen in tissues exposed to the calcium ionophore A 23187 (1 μm). In calcium depleted tissues, cyclopiazonic acid (3 μm) prevented restoration of the cis‐dioxolane‐induced contraction following re‐exposure to a calcium containing high K+ buffer, but failed to inhibit the photoresponse. 5 Both the fast and slow relaxations were potentiated by zaprinast (10 μm) and inhibited by LY 83583 (10 μm). However, in calcium‐depleted, calyculin A‐precontracted preparations only the slow relaxation was evident. 6 The present results support the conclusion that: (i) functional L‐channels are required for the expression of the fast components of the 3‐NO2‐DHP‐ or NO‐donor‐induced photoresponse, (ii) NO photorelease followed by activation of soluble guanylyl cyclase is responsible for the photosensitizing activity of 3‐NO2‐DHPs and (iii) regulation of the contractile proteins via cyclic GMP‐dependent phosphorylation may underlie the slow relaxation.

Brainstem Control of Deglutition: Brainstem Neural Circuits and Mediators Regulating Swallowing

Swallowing requires coordination of several paired muscle groups in the head and neck including the diaphragm. Thus, motoneurons of the Vth, VIIth, IX through XIIth cranial, cervical spinal, and phrenic nerves are sequentially activated by a swallowing pattern generator (SPG) located in the lower brainstem for executing the oral, pharyngeal, and esophageal stages of swallowing. Independence of these stages from each other indicates distinct subcircuits, their coupling pointing to flexible links between them. Although intrinsically autonomous, the SPG depends on peripheral and suprabulbar afferents for proper functioning. Pivotal to both, integrating afferents and coordinating stage-specific subcircuits, are the nucleus tractus solitarii (NTS) with some of its subnuclei and their reciprocal interconnections with the brainstem reticular formation. This chapter provides a survey of the anatomical and functional organization of the SPG.

Neurogenic responses in rat and porcine large pulmonary arteries

Pharmacological differences between neurogenic sympathetic responses in rat and pig isolated pulmonary arteries were examined in strip preparations. Electrical field stimulation in the range of 0.6 to 40 Hz resulted in frequency-dependent contractions in terms of amplitude and rate of rise. Responses in the rat declined sharply from pulmonary trunk to main artery; in contrast, in the pig they continued into the third-order vessels. Contractions were inhibited in the presence of tetrodotoxin, prazosin or WB-4101 and hence neurogenic in origin. Cocaine enhanced field stimulated contractions in both rat and porcine tissues; however, the effect in the former was of significantly greater magnitude in terms of either area under the mechanogram or height of contraction. In addition, the rate of rise, time to peak and duration of peak were all increased in the rat but less so or not in the pig. Field stimulated contractions were virtually abolished by guanethidine (1×10−6 M) in rat but not in porcine pulmonary arteries in which a ten-fold higher concentration significantly reduced neurogenic contractions and abolished them in 2 out of 4 tissues tested. The effect of guanethidine (1×10−6 M) observed in blood vessels of rat exceeded about five-fold that observed in porcine tissues. Thus, neurogenic responses appear to be entirely mediated by extra-junctional α1-adrenoceptors in both species, and in contrast to the rat, pig tissues seem to have a noradrenaline re-uptake that is either less efficient or operating near saturation.

Role of Guanylate Cyclase Activation in the Smooth Muscle Actions of Dihydropyridines

Certain 1,4-dihydropyridines (DHPs) interact with the ‘L’-type voltage-operated Ca2+ channel (VOCC) and thereby modulate Ca2+ entry into a variety of excitable tissues. Radioligand binding data indicates that the DHP nifedipine binds with high affinity to the VOCC and electrophysiological data indicates that a Ca2+ current can be modulated by such compounds1. The high affinity that many DHPs show for the VOCC is suggestive that other potential sites of action are of secondary importance with respect to their effects on smooth muscle function. It is thus of interest to note that comparatively low affinity sites have been described for the organic Ca2+ channel modulators2. Although such sites are of low affinity it must be emphasized that since the organic Ca2+ channel antagonists are hydrophobic drugs, they are capable of accumulating within vascular smooth muscle cells at tissue:medium ratios of 200–500:13.

Transmural pressure and membrane potential in human saphenous vein

OBJECTIVE An increase in transmural pressure reportedly depolarizes myocytes in various arterial blood vessels. We have examined the relationship between transmural pressure and membrane potential (E(m)) in human saphenous veins with a view to determine whether contractile force generation, hence spasmogenesis in vein grafts, involves a similar process of mechanoelectrical excitation. METHODS Intracellular recordings were made by sharp glass microelectrodes in human isolated saphenous veins and parallel measurements were performed in ring preparations. RESULTS E(m) values obtained in pressurized vessels at four different pressure levels were (mean+/-SD): -74.4+/-5.5 mV (0-6 cm H(2)O; n=10), -72.6+/-6.5 mV (11-14 cm H(2)O; n=27), -72.1+/-6.5 mV (26-27 cm H(2)O; n=30), and -72.9+/-4.0 mV (50-54 cm H(2)O; n=38), demonstrating the lack of an overt pressure-dependence. Except at the lowest transmural pressure tested, these values were significantly different from E(m) obtained in ring preparations (-77.8+/-4.0 mV; n=30). Raising extracellular K(+) to 80 mM produced a comparable depolarization in tissues either pressurized to 50-54 cm H(2)O (-64.9+/-4.3 mV; n=27) or set up as ring preparations (-64.06+/-6.9 mV; n=35). CONCLUSIONS Human saphenous veins respond to transmural pressure with a limited depolarization that lacks correlation with pressure. The absence of a pressure-induced graded depolarization suggests that pressure-dependent vasoconstriction does not play a primary role in blood flow regulation in lower limb large veins. Moreover, this raises doubts that mechanical stimuli per se would lead to development of vasospasm in the early stages of saphenous vein grafting into arterial vascular beds.