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D J Bennett

Publications and source records attributed to D J Bennett.

At least 37 records · Page 2Linked to original sources
PubMed1998

Self-sustained firing of human motor units.

Motoneurons of invertebrates and vertebrates can continue to fire repetitively after being activated by a brief, excitatory synaptic input (self-sustained firing). This firing behavior is due to the activation of intrinsic, voltage-gated currents which produce sustained regenerative depolarizations (plateau potentials) of the cell. Examination of these intrinsic cellular properties has been performed in reduced animal preparations and it is unknown if such self-sustained firing occurs in motoneurons of the intact human. In this paper, we present evidence of this in the human by using a technique of dual motor unit recordings. Subjects were instructed to maintain a constant dorsiflexion effort, and the common synaptic input (e.g. descending drive) onto the tibialis anterior (TA) motoneuron pool was monitored by recording the firing frequency of a low threshold 'control' unit. Once the firing rate of the control unit was constant, vibration of the TA tendon recruited a second 'test' unit which continued to fire after the vibration (i.e. synaptic input) was removed, even though the firing rate of the control unit (and thus, the common drive) remained the same or decreased. Self-sustained firing of motoneurons such as this may reduce the need for prolonged synaptic input when constant muscle activation is required (e.g. for postural tone).

Evoked Potentials, Motor↗
PubMed1998

Effect of sensory denervation on the structure and physiologic responsiveness of rabbit lacrimal gland.

PURPOSE: This work was conducted to determine the effects of unilateral trigeminal ganglion ablation on lacrimal gland structure and secretory activity. METHODS: Adult male New Zealand rabbits underwent unilateral thermocoagulation of the ophthalmic division of the trigeminal ganglion. Sensory denervation was affirmed by anatomic inspection of the lesion and transmission electron microscopy (TEM) of the lacrimal gland innervation. Eight to 10 days after the procedure, the intraorbital lacrimal glands were removed from both sides. To compare the physiologic competence of the intact and denervated glands, freshly isolated gland fragments from the paired intact and denervated glands were stimulated with carbachol (100 microM), isoproterenol (10 microM), phorbol-12,13-dibutyrate (PDBu, 10 microM), forskolin (40 microM), or vehicle. Total secreted protein was measured at 30 or 60 min after the establishment of baseline values. Intact and denervated glands also were examined by light and TEM, and the morphologic appearance of the acinar structures as well as the appearance of nerves innervating the gland after denervation were assessed. Similar experiments were conducted with animals that underwent unilateral superior cervical ganglionectomy. RESULTS: Tissues from sensory denervated glands released significantly more protein than did tissues from innervated glands in response to in vitro stimulation by carbachol or isoproterenol but not in response to PDBu or forskolin. Microscopy showed that the acinar cells that had undergone sensory denervation showed a massive accumulation of secretory granules. The secretory granules filled the entire cytoplasmic space and displaced the ellipsoidal nuclei to the extreme periphery. Examination of segments of nerves revealed numerous unmyelinated axons, a few small-diameter myelinated axons, and a large amount of nerve degeneration after sensory denervation. In contrast to the effects of sensory denervation, sympathetic denervation did not alter either the acinar appearance or secretory responsiveness of the gland. CONCLUSION: Loss of the considerable sensory innervation from the trigeminal ganglion has pronounced effects on the pharmacologic responsiveness and the structure of the lacrimal gland. The effects of sensory innervation on the gland may be mediated through two possible pathways: direct input to the gland or control of the preganglionic parasympathetic pathway.

Animals↗
PubMed1998

Synaptic activation of plateaus in hindlimb motoneurons of decerebrate cats.

Intracellular recordings were made from hindlimb motoneurons in decerebrate cats to study how synaptic inputs could affect the threshold at which plateau potentials are activated with current injections through the recording microelectrode in the cell body. This study was prompted by recent evidence that the noninactivating inward currents that regeneratively produce the plateau potentials arise (partly) from dendritic conductances, which may be relatively more accessible to synaptic input than to current injected into the soma. Initially, cells were studied by injecting a slow triangular current ramp intracellularly to determine the threshold for activation of the plateau. In cells where the sodium spikes were blocked with intracellular QX314, plateau activation was readily seen as a sudden jump in membrane potential, which was not directly reversed as the current was decreased. With normal spiking, the plateau activation (the noninactivating inward current) was reflected by a steep and sustained jump in firing rate that was not directly reversed as the current was decreased. Importantly, the threshold for plateau activation (at 34 Hz on average) was significantly above the recruitment level (13 Hz on average). When tonic synaptic excitation [excitatory postsynaptic potentials (EPSPs)] was provided either by stretching the triceps surae muscle or by stimulating its nerve at a high frequency, the threshold for plateau activation by intracellular current injection was significantly lowered (by 12 Hz or 5.8 mV on average, without and with QX314, respectively). Conversely, tonic synaptic inhibition [inhibitory postsynaptic potentials (IPSPs)], provided by appropriate nerve stimulation, significantly raised the plateau threshold (by 19 Hz or 7.6 mV on average). These effects were graded with the intensity of tonic EPSPs and IPSPs. Strong enough EPSPs brought the plateau threshold down sufficiently that it was activated by the intracellular current soon after recruitment. A further increase in tonic EPSPs recruited the cell directly, and in this case the plateau was activated at or before recruitment. The finding that synaptic excitation can produce plateau activation below the recruitment level is of importance for the interpretation of its function. With this low-threshold activation, the plateau potentials are likely important in securing an effective recruitment to frequencies that produce significant force generation and would subsequently have no further affect on the frequency modulation, other than to provide a steady depolarizing bias that would help to sustain firing (cf. self-sustained firing). Additional jumps in frequency after recruitment (i.e., bistable firing) would not be expected.

Action Potentials↗
PubMed1998

Short-term plasticity in hindlimb motoneurons of decerebrate cats.

Cat hindlimb motoneurons possess noninactivating voltage-gated inward currents that can, under appropriate conditions, regeneratively produce sustained increments in depolarization and firing of the cell (i.e., plateau potentials). Recent studies in turtle dorsal horn neurons and motoneurons indicate that facilitation of plateaus occurs with repeated plateau activation (decreased threshold and increased duration; this phenomenon is referred to as warm-up). The purpose of the present study was to study warm-up in cat motoneurons. Initially, cells were studied by injecting a slow triangular current ramp intracellularly to determine the threshold for activation of the plateau. In cells where the sodium spikes were blocked with intracellular QX314, plateau activation was readily seen as a sudden jump in membrane potential, which was not directly reversed as the current was decreased (cf. hysteresis). With normal spiking, the plateau activation (the noninactivating inward current) was reflected by a steep and sustained jump in firing rate, which was not directly reversed as the current was decreased (hysteresis). Repetitive plateau activation significantly lowered the plateau activation threshold in 83% of cells (by on average 5 mV and 11 Hz with and without QX314, respectively). This interaction between successive plateaus (warm-up) occurred when tested with 3- to 6-s intervals; no interaction occurred at times >20 s. Plateaus initiated by synaptic activation from muscle stretch were also facilitated by repetition. Repeated slow muscle stretches that produced small phasic responses when a cell was hyperpolarized with intracellular current bias produced a larger and more prolonged responses (plateau) when the bias was removed, and the amplitude and duration of this response grew with repetition. The effects of warm-up seen with intracellular recordings during muscle stretch could also be recorded extracellularly with gross electromyographic (EMG) recordings. That is, the same repetitive stretch as above produced a progressively larger and more prolonged EMG response. Warm-up may be a functionally important form of short-term plasticity in motoneurons that secures efficient motor output once a threshold level is reached for a significant period. Finally, the finding that warm-up can be readily observed with gross EMG recordings will be useful in future studies of plateaus in awake animals and humans.

Animals↗
PubMed1998

Gi2 and Gi3 couple met-enkephalin to inhibition of lacrimal secretion.

PURPOSE: The intent of this study was to identify the pertussis toxin-sensitive G proteins that couple met-enkephalin to the inhibition of cholinergically stimulated secretion in rabbit lacrimal gland acini. METHODS: The authors detected G proteins in membranes from freshly isolated glands, freshly isolated acini, and cultured lacrimal acini from rabbits by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting. Antibodies against the alpha subunits of Gi1, Gi1 and Gi2, or Gi3 were used in cultured acini permeabilized by streptolysin-O to determine the role of the G proteins in met-enkephalin inhibition of cholinergic stimulation of lacrimal acinar protein release. RESULTS: Western blot analysis showed the presence of the alpha subunits of Gi2 and Gi3, but not Gi1, in all three membrane preparations. The met-enkephalin analog D-Ala2-methionine enkephalinamide (DALA) inhibited cholinergic stimulation of secretion by cultured rabbit acinar cells to near basal levels. Inhibition of secretion by DALA was blocked by insertion of antibody to a peptide sequence common to Gialpha1 and Gialpha2, but was not blocked by antibody against a specific Gialpha1 sequence. The inhibitory effect of DALA also was blocked by antibody to a Gialpha3 sequence. At low doses of anti-Gialpha1/2 and anti-Gialpha3 in combination, the effect on reversal of inhibition was additive. However, at higher doses, the effect of the combination was no greater than the effect of either antibody alone. CONCLUSIONS: These results demonstrate that met-enkephalin inhibition of cholinergic secretion is mediated by way of the pertussis toxin-sensitive G proteins Gi2 and Gi3 in cultured rabbit lacrimal acini. Because the effects of the G proteins are not additive, the intracellular events distal to G protein activation most likely converge at some point before exocytosis.

Animals↗
PubMed1997

Measurement of rigidity in Parkinson's disease.

Clinical assessment of rigidity in parkinsonian patients is largely qualitative. The reliability and validity of the assessments are sometimes in doubt. Several "engineering" methods of quantifying rigidity have been described, but none has been adopted into general clinical practice. A possible reason is that these methods differ in crucial aspects from the clinical exam. We therefore tackled the problem by monitoring the clinical exam itself, using small sensors to measure the forces and displacements applied. Limb impedance (Z) was computed using parameter identification methods and compared to raters' verbalized ratings of rigidity based on a 5-point scale: the Unified Parkinson's Disease Rating System. The qualitative and quantitative estimates of impedance covaried over a fourfold range, depending on the forces imposed and the subject's motor set. Raters differed by up to 1 full point in their mean qualitative ratings and sometimes disagreed on whether levodopa reduced rigidity. This was not due to any significant differences in the overall range of rigidity they evoked, but rather to the way they scored this range [the ratio of mean rating to mean impedance (R/Z) varied between raters and subjects]. On the other hand, the R/Z ratio was reproducible over separate sets of ratings and may therefore serve to convert measured impedance into a standardized rating. Our results indicate that the current clinical exam may be too abbreviated to detect the sometimes quite small reductions in rigidity after levodopa. We conclude that a device that conveniently quantifies the clinical assessment of rigidity is now available and will lead to more standardized protocols for rating rigidity in the near future.

Antiparkinson Agents↗
PubMed1997

Recollective and automatic uses of memory.

Three cued-recall experiments examined the effects of learning conditions and set size on recollective and automatic uses of memory. Words were studied under different conditions and had either small or large preexisting associative sets. Results based on the process-dissociation procedure showed that learning conditions and set size influenced recollective uses of memory, whereas only set size influenced automatic uses. Other results indicated that process-dissociation and direct-indirect test procedures produce similar results. The findings suggest that the relative contributions of recently acquired information and preexisting information depend on how memory is being used. Recollective uses of memory are affected by the nature of recent study and by what such study activates in long-term memory. In contrast, automatic uses of memory are more affected by what the test cue automatically activates than by what has been learned during recent study.

Automatism↗
PubMed1997

Positive force feedback control of muscles.

This study was prompted by recent evidence for the existence of positive force feedback in feline locomotor control. Our aim was to establish some basic properties of positive force feedback in relation to load compensation, stability, intrinsic muscle properties, and interaction with displacement feedback. In human subjects, muscles acting about the wrist and ankle were activated by feedback-controlled electrical stimulation. The feedback signals were obtained from sensors monitoring force and displacement. The signals were filtered to mimic transduction by mammalian tendon organ and muscle spindle receptors. We found that when muscles under positive force feedback were loaded inertially, they responded in a stable manner with increased active force. The activation attenuated the muscle stretch (yield) that would otherwise occur in the absence of feedback. With enough positive force feedback gain, yield could actually reverse. This behavior, which we termed the affirming reaction, was reminiscent of the mammalian positive supporting reaction, a postural response elicited by contact of the foot with the ground. Muscles under positive force feedback remained stable, even when the loop gain (Gf) was set at levels of 2 or 3. In a linear system, if Gf > 1, instability occurs when the loop is closed. On further investigation, we found that Gf changed with joint angle: it declined as the load-bearing muscle actively shortened. We inferred that in closed-loop operation, the active muscles always shortened until Gf approached unity. In other words, the length-tension curve of active muscle ensures stability even when force-related excitation of motoneurons is very large. Concomitant negative displacement feedback reinforced and stabilized load compensation up to a certain gain, beyond which instability occurred. In further trials we included delays of up to 40 ms in the positive force feedback pathway, to model the delays recently described for tendon organ reflexes in cat locomotion. Contrary to expectations, this did not destabilize the loop. Indeed, when instability was deliberately evoked by setting displacement feedback gain high, delays in the positive force feedback pathway actually stabilized control. The stabilization of positive force feedback by inherent properties of the neuromuscular system increases the functional scope to be expected of feedback from force receptors in biological motor control. Our results provide a rationale for the delayed excitatory action of Ib heteronymous input on extensor motoneurons in cat locomotion.

Adult↗
PubMed1997

Implications of positive feedback in the control of movement.

In this paper we review some theoretical aspects of positive feedback in the control of movement. The focus is mainly on new theories regarding the reflexive role of sensory signals from mammalian tendon organ afferents. In static postures these afferents generally mediate negative force feedback. But in locomotion there is evidence of a switch to positive force feedback action. Positive feedback is often associated with instability and oscillation, neither of which occur in normal locomotion. We address this paradox with the use of analytic models of the neuromuscular control system. It is shown that positive force feedback contributes to load compensation and is surprisingly stable because the length-tension properties of mammalian muscle provide automatic gain control. This mechanism can stabilize control even when positive feedback is very strong. The models also show how positive force feedback is stabilized by concomitant negative displacement feedback and, unexpectedly, by delays in the positive feedback pathway. Other examples of positive feedback in animal motor control systems are discussed, including the beta-fusimotor system, which mediates positive feedback of displacement. In general it is seen that positive feedback reduces the sensitivity of the controlled extremities to perturbations of posture and load. We conclude that positive force feedback can provide stable and effective load compensation that complements the action of negative displacement and velocity feedback.

Afferent Pathways↗
PubMed1997

One step is not enough: making better use of association norms to predict cued recall.

Cued recall is strongly affected by the strength of the preexisting connection between the test cue and the information to be recalled, the target. In all past work, preexisting cue-to-target strength has been measured by the probability that the cue produced the target in free association. This paper presents four experiments showing that this use of such norms underestimates the strength of the connection and that a more accurate estimate can be obtained by incorporating indirect as well as direct connections in the estimate. Experiments 1 and 2 showed that in extralist cued recall both the strength and number of two-step indirect connections facilitate recall. Experiment 3 showed that three-step connections have negligible effects. Experiment 4 used an intralist task in which cue and target are first studied together, and the results showed once again that indirect connections can affect recall. In all of these experiments, indirect connections had an effect on recall that was larger when direct cue-to-target strength was weak than when it was strong. Implications for using association norms in research are described, and an algorithm for using association norms to measure cue-to-target strength is proposed.

Adult↗
PubMed1997

Gs and Gq/11 couple vasoactive intestinal peptide and cholinergic stimulation to lacrimal secretion.

PURPOSE: The intent of this study was to determine the physiological role of selected G proteins in receptor-mediated protein release by lacrimal acini. METHODS: The role of G proteins in lacrimal secretion was determined in tissues obtained from the lacrimal glands of adult male New Zealand White rabbits. Pertussis toxin treatment of primary acinar cultures and permeabilization of cultured acini with streptolysin-O and insertion of GDP beta S or antibodies against the alpha subunit of Gs or Gq/11 were used to determine the role of G proteins in vasoactive intestinal peptide (VIP) and carbachol-stimulated lacrimal secretion. Gs and Gq/11 were identified in lacrimal membranes obtained from freshly isolated lacrimal gland fragments, freshly isolated acini, and cultured acini by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting. RESULTS: Permeabilization by streptolysin-O and introduction of guanosine thiodiphosphate into cultured acini blocked stimulation of protein released by either 100 nM VIP or 100 microM carbachol by approximately 50%. Exposure of cultured acini to 100 ng/ml pertussis toxin for 36 to 48 hours did not affect stimulated release by either agonist, indicating that the guanosine triphosphate-dependent actions of VIP and carbachol are mediated through pertussis toxin-insensitive G proteins. Pertussis toxin-insensitive G proteins in lacrimal membranes obtained from freshly isolated glands, freshly isolated acini, and cultured acini were identified with polyclonal antibodies to the alpha subunits of Gs and Gq/11. Immunoblotting of lacrimal membranes with anti-Gs alpha antiserum showed two immunoreactive bands at 44 and 47 kDa. Anti-Gq/11 alpha antiserum detected a single band at 46 kDa in similar membrane preparations. Anti-Gs alpha antiserum reduced the secretory response to VIP by 64% and to carbachol by 37%. Introduction of anti-Gq/11 alpha antiserum reduced the response to carbachol by 70%; however, the response to VIP was unchanged. Simultaneous introduction of both antisera caused no further reduction of VIP-stimulated release than did anti-Gs alpha antiserum alone. However, simultaneous introduction of both anti-Gs alpha and anti-Gq/11 alpha antisera resulted in complete inhibition of the effects of carbachol on protein release by cultured acini. CONCLUSIONS: These results show that VIP receptor activation of lacrimal protein release is mediated through Gs, whereas cholinergic stimulation involves both Gs and Gq/11. From the authors' results, the authors conclude that Gs links VIP receptor activation to adenylyl cyclase and cyclic adenosine 3'-5' monophosphate production and the ultimate release of protein by acinar cells and that Gq/11 links muscarinic receptor activation to phospholipase C and IP3 and diacylglycerol accumulation, which also leads to protein release. Furthermore, it is hypothesized that Gs has an additional role in the regulation of vesicular traffic and exocytosis.

Adenylate Cyclase Toxin↗
PubMed1996

Gain of the triceps surae stretch reflex in decerebrate and spinal cats during postural and locomotor activities.

1. The triceps surae (TS) stretch reflex was measured in decerebrate cats during crossed extensor stimulation and after spinalization during rhythmic locomotor activity induced by clonidine and manual perineal stimulation. The TS force in response to sinusoidal stretch was measured at a given contraction level before and after deafferentation, and the 'reflex force' was computed by subtracting these two responses. Reflex 'gain' was computed as the ratio of the reflex and deafferented force responses (a unitless estimate of the open loop feedback gain). 2. Prior to locomotion the spontaneous muscle activity was low (less than 15% of maximum), but the reflex gain was relatively high (close to 1.0 with a 5 Hz stretch). When locomotion commenced the reflex gain was markedly lowered when measured at the same contraction level as before locomotion (25% of the gain prior to locomotion). At higher contraction levels the reflex gain was not significantly increased. The reflex force and EMG responses to stretch increased with the contraction level, but their effect on the total reflex gain was cancelled by an associated increase in the intrinsic muscle stiffness. 3. In the decerebrate cat, during weak tonic contractions (spontaneous), the reflex gain was high and comparable with the gain in the resting spinal cat. However, with increased tonic contractions produced by crossed extensor stimulation the reflex gain dropped. At higher contraction levels the gain was not significantly different from the gain during spinal locomotion. 4. When the frequency of stretch was increased from 3 to 20 Hz, EMG responses to stretch increased, but the reflex force decreased, since a more fused contraction developed with the more frequent reflex activations. Overall, the reflex gain decreased with frequency in both spinal and decerebrate cats. The phase lag of the reflex force, relative to the intrinsic muscle force, increased with increasing frequency, due to reflex delays, with a 180 deg lag occurring between 12 and 18 Hz (tremor frequencies). The mean gain was significantly lower and the phase lag was significantly greater during locomotion than during tonic crossed extensor contractions, suggesting different reflex mechanisms. 5. In conclusion, during locomotion in spinal cats afferent feedback from low frequency ankle movements, similar to those occurring during the normal step cycle, reflexly produces a small but significant fraction of the extensor force (about a quarter of the stretch-related force modulation). This fraction is remarkably constant at the different contraction levels of the step cycle. Afferent feedback during higher frequency movement is less effective, minimizing the chance of instability and tremor. In contrast during tonic contractions afferent feedback produces half of the total muscle force during perturbations, clearly contributing to the maintenance of posture.

Afferent Pathways↗
PubMed1996

Regulation of soleus muscle spindle sensitivity in decerebrate and spinal cats during postural and locomotor activities.

1. In order to study fusimotor control in reduced preparations, soleus muscle spindle afferents were recorded in premammillary decerebrate cats (n = 15) during crossed extensor reflexes and, after spinalization, during locomotion produced by either clonidine or L-beta-3,4-dihydroxyphenylalanine (L-DOPA). The soleus muscle was oscillated sinusoidally (0.25 mm, 4 Hz) and the afferent mean firing rate and modulation were calculated. An increase in firing rate was assumed to arise from activity in dynamic gamma-motoneurones (dynamic gamma-drive) when associated with an increase in modulation to stretching, and in static gamma-motoneurones (static gamma-drive) when modulation decreased. 2. At rest in all preparations the firing rate and modulation in primary muscle spindle afferents were generally much higher than after de-efferentation (ventral root section), suggesting a predominant dynamic gamma-drive. Clonidine decreased and even eliminated this presumed resting gamma-drive in many afferents, both in the decerebrate (7 of 8) and the spinal (6 of 18) state. This effect on gamma-drive may account, at least in part, for its suppressive effect on spasticity in humans. 3. When locomotion commenced in clonidine-treated spinal cats, primary afferents generally fired with much higher mean rates (+121%) and lower sensitivities (-32%), suggesting a large increase in static gamma-drive (possibly accompanied by a small decrease in dynamic gamma-drive). These high rates were usually maintained tonically throughout the step cycle. However, a third of the afferents were silenced during locomotor contractions, and de-efferentation had no significant effect on their firing rates. Thus, for some spindles alpha-activity can occur without significant gamma-drive. 4. During locomotion in L-DOPA-treated spinal cats the inferred static gamma-drive only occurred phasically, coactivated with the EMG, though it could precede the EMG by 100-500 ms. In the flexion phase both the afferent rate and modulation were lower than before locomotion, suggesting a lack of effective gamma-drive. 5. Crossed extensor reflexes in decerebrate cats also produced a substantial increase in primary afferent firing rate (+187%) and decrease in sensitivity (-37%), again suggesting increased static gamma-drive (n = 18). This gamma-drive was largely independent of EMG activity and often occurred without alpha-activity. The mean firing rate of secondary muscle spindle afferents increased significantly during locomotion (with L-DOPA) and crossed extensor reflexes, again indicating increased static gamma-drive. Clonidine reduced or eliminated the gamma-drive in seven of eight afferents during crossed extensor reflexes. 6. In conclusion, although there are some common features, such as a predominant static gamma-drive in all walking preparations, the pattern of static and dynamic gamma-drive is not closely linked to alpha-activity under the conditions studied. As well as gamma-drive without alpha-activity, we have shown for the first time that alpha-motoneurones can be activated without significant gamma-drive to many spindles during behavioural tasks.

Action Potentials↗
PubMed1995

Piperazinylalkyl heterocycles as potential antipsychotic agents.

We recently reported on a series of pyrrole Mannich bases orally active in inhibiting the conditioned avoidance response (CAR) in rats. These compounds exhibit affinity for both D2 and 5-HT1A receptors, and some are noncataleptogenic. Such a profile suggests that they may be potential antipsychotic agents which lack the propensity for causing extrapyramidal side effects and tardive dyskinesias in humans. One of these compounds, 1-[[1-methyl-5-[[4-[2-(1-methylethoxy)phenyl]- 1-piperazinyl]methyl]-1H-pyrrol-2-yl]methyl]-2-piperidinone (RWJ 25730, 1), was chosen for further development but found to be unstable in dilute acid. In order to improve stability, we replaced the pyrrole methylene linkage to the piperazine ring with ethylene, employed ethylene and dicarbonyl as linkers between the lactam and the pyrrole ring, placed electron-withdrawing groups on the pyrrole ring, and substituted acyclic amide for lactam. In addition, we replaced the pyrrole segment with other heterocycles including thiophene, furan, isoxazole, isoxazoline, and pyridine. Generally, replacement of the N-methylpyrrole segment with thiophene, furan, isoxazoline, or pyridine afforded compounds equipotent with 1 in CAR, which were more stable in dilute acid. In the case of side chain or lactam modifications, CAR activity was significantly decreased or abolished, with the exception of 6. For the most part, the modifications to 1 resulted in the decrease or loss of D2 receptor binding. However, within this series, 5-HT1A receptor binding was greatly increased, with thiophene 40 exhibiting an IC50 of 0.07 nM. The CAR activities of pyrroles 6 and 12, thiophene 40, furans 44-47, isoxazolines 49 and 50, and pyridine 54 coupled with their weak or nonexistent D2 binding and strong 5-HT1A binding suggest that they may be acting via a nondopaminergic mechanism or that dopaminergic active metabolites are responsible. Pyrrole 6 and furans 44 and 47 show promise as antipsychotic agents based on their CAR activity, receptor-binding profile, and solution stability.

Animals↗
PubMed1995

N-aryl-N'-benzylpiperazines as potential antipsychotic agents.

N1-(2-Alkoxyphenyl)piperazines additionally containing an N4-benzyl group bearing alcohol, amide, imide, or hydantoin functionalities were prepared and evaluated in the conditioned avoidance response (CAR) test predictive of clinical antipsychotic activity and in in vitro receptor-binding assays. Certain of the compounds display high affinity for the D2, 5-HT1A, and alpha 1-adrenergic receptors. Structures bearing acyclic amide, lactam, and imide functionalities display good biological activity, with a preference for the 1,3-disubstituted phenyl ring relative to the 1,4- and 1,2-congeners (7 vs 10 and 12). Every possible position of hydantoin attachment was investigated (e.g., substitution at N1, N3, and C5). The hydantoin involving attachment to N1 (24) was found to have good biological activity, whereas those hydantoins with attachment to N3 or C5 (22, 23, and 25) were inactive. Several of the smaller acetylated derivatives (30 and 33) have fair in vivo activity, which was lost in the case of the larger benzoyl analog 31. Uracil congener 34 had modest affinity for the D2 receptor (65 nM) as well as excellent in vivo activity. Benzylamino compounds display (viz. 27 and 35-38) moderate CAR activity but have surprising receptor affinity, often greater than those of comparable structures bearing a carbonyl (36 vs 7). Benzyl and benzhydryl alcohol compounds 40-48 are more active than amino structures 27 and 35-38 and also exhibit excellent in vivo activity in the CAR test with modest D2 and 5-HT1A receptor binding.

Animals↗
PubMed1995

Identification of G proteins in lacrimal gland.

PURPOSE: The intent of this study was to identify and characterize guanine nucleotide binding proteins (G proteins) that are a component of cell signaling of stimulus-secretion coupling in lacrimal gland. METHODS: Membranes were isolated from the lacrimal glands of male Sprague-Dawley rats and New Zealand rabbits and were used to identify G proteins in lacrimal gland by Western blot analysis. Solubilized membrane proteins from lacrimal glands and from a positive tissue control (SH-SY5Y cells) were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The alpha subunits of the G proteins were identified by immunoreactivity, with specific peptide directed antisera against Gs alpha, Gi alpha 1, Gi alpha 1/2, and Go alpha. The initial characterization of coupling of specific G proteins to receptors was accomplished by preincubation of membranes with nonimmune serum, anti-Gs alpha, or anti-Go alpha antiserum and assay of adenylyl cyclase activity in the presence of the neuropeptide, vasoactive intestinal peptide (VIP), or forskolin. RESULTS: Gs alpha, Gi alpha 3, and Go alpha were present in all three membrane preparations. Gs alpha subunits were detected as two bands at 45 to 48 kd and 48 to 54 kd. Gi alpha 3 was detected as a single protein at 40 to 41 kd, and at least one form of Go alpha with a molecular weight of 40 kd was detected in all three preparations. Gi alpha 1 was detected in immunoblots of rat lacrimal and SH-SY5Y membranes at 41 kd, and the density of the band was enhanced in blots probed with anti-Gi alpha 1/2 antiserum. Immunoreactivity to anti-Gi alpha 1 or anti-Gi alpha 1/2 was faint or not detectable in rabbit lacrimal membranes. A prominent band was detected in rabbit and rat lacrimal but not in SH-SY5Y membranes at 31 kd with anti-Gi alpha 1/2 antiserum, which may represent a G protein involved in exocytosis. Coupling of VIP receptor activation to adenylyl cyclase by Gs alpha was evidenced by the reduction of VIP stimulation of the enzyme by preincubation of rabbit membranes with anti-Gs alpha antiserum. In contrast, preincubation of membranes with anti-Go alpha antiserum resulted in an increase in activity of adenylyl cyclase in the presence of VIP. CONCLUSIONS: Detection of specific alpha subunits in lacrimal gland indicates that Gs, Gi, and Go are present in rabbit and rat lacrimal gland. Both Gs and Go influence VIP stimulation of lacrimal adenylyl cyclase and thus are presumed to be involved in signal transduction, leading to second-messenger accumulation and subsequent regulation of lacriminal function, including secretion. In addition, an unidentified protein is present in lacrimal gland that may represent one of the guanine nucleotide binding proteins involved in exocytosis.

Adenylyl Cyclases↗