Integrating medical specialists and hospitals. The growing relevance of collective organisation of medical specialists for Dutch hospital governance.

The policy of the Dutch government (now enshrined in legislation) aims at the integration of medical specialists in hospitals and seeks to end the economic and organisational autonomy of the medical specialist. This article suggests that this policy has also acted as an incentive for medical specialists to develop local strategies that often diverge. Medical specialists are pursuing strategies that aim to strengthen their collective power within the hospital organisation while at the same time consolidating their entrepreneurial status. Medical staff governance has consequently become a key factor in hospital governance.

Sequential changes in synaptic vesicle pools and endosome-like organelles during depolarization near the active zone of central nerve terminals.

During periods of high-frequency stimulation the maintenance of synaptic transmission depends on a continued supply of synaptic vesicles. Local recycling in the terminals ensures synaptic vesicle replenishment, but the intermediate steps are still a matter of debate. We analyzed changes in synaptic vesicle pools and endosome-like organelles near the active zone in central nerve terminals during depolarization at the ultrastructural level by electron microscopy. A short, 100 ms, depolarization-induced recruitment of synaptic vesicles was observed from a reserve pool to a recruited pool, within 150 nm of the active zone, and the docked pool at the active zone was increased as well. Prolonged, 15 s or 3 min, depolarization decreased the total amount of synaptic vesicles, which was accompanied by a parallel increase in size and amount of endosome-like organelles. After a period of rest, the number of endosome-like organelles decreased and the amount of synaptic vesicles was restored to control level. The endocytotic nature of part of the endosome-like organelles after 15 s and 3 min depolarization was indicated by their labeling with extracellularly added horseradish peroxidase (HRP). In addition, a small number of synaptic vesicles entrapped HRP under these conditions. After repolarization, the number of HRP-loaded endosome-like structures decreased. Simultaneously, a strong increase in amount of HRP-loaded small vesicles did occur. These results indicate that during sub-second depolarization, synaptic vesicles were rapidly recruited from the reserve pool to replenish the releasable pool, whereas prolonged depolarization (s-min) induced local endocytosis in at least two ways, i.e. either directly as vesicles or via endosome-like organelles from which synaptic vesicles were reformed.

Activity-dependent neurotransmitter release kinetics: correlation with changes in morphological distributions of small and large vesicles in central nerve terminals.

In central nerve terminals transmitter release is tightly regulated and thought to occur in a number of steps. These steps include vesicle mobilization and docking prior to neurotransmitter release. Intrasynaptic changes in vesicle distribution were determined by electron microscopical analysis and neurotransmitter release was monitored by biochemical measurements. We correlated K + -induced changes in distribution of small and large vesicles with the release of their transmitters. For small synaptic vesicles, amino acid release as well as recruitment to and docking at the active zone were activated within 1 s of depolarization. In contrast, the disappearance of large dense-cored vesicles and the release of the neuropeptide cholecystokinin were much slower, and no docking was observed. Studies with diverse Ca2 + channel blockers indicated that mobilization and neurotransmitter release from both vesicle types were regulated by multiple Ca2 + channels, although in different ways. Neurotransmitter release from small synaptic vesicles was predominantly regulated by P-type Ca2 + channels, whereas primarily Q-type Ca2 + channels regulated neurotransmitter release from large dense-cored vesicles. The different Ca2 + channnel types directly regulated mobilization of and neurotransmitter release from small synaptic vesicles whereas, by their cooperativity in raising the intracellular Ca2 + concentration above release threshold, they more indirectly regulated large dense-cored vesicle exocytosis.

Between physician and manager: new co-operation models in Dutch hospitals.

Analyses the way hospital organisation models handle the relationship between medical specialists and hospital management. All models that have been developed during the last ten years seek to integrate the medical specialists in the hospital organisation by formally subordinating them to the hospital management. However, recently a new model has come to the fore--the "co-makership"--in which the hospital management and the medical specialists are assigned a position alongside each other.

Cost-control and medical specialist payment: the Dutch alternative.

The fee-for-service system is a growing problem for insurers and governments. The main reason for this is the open-ended character of this system which makes cost-control a very difficult task. The pressures on the fee-for-service system are becoming more pronounced, especially in countries such as Canada, Germany and the Netherlands which use budget restrictions on national health care expenditure (macro caps). In these countries policy makers are searching for an alternative payment system and an appropriate definition of a corresponding status for doctors. The alternative, however, does not have to lead automatically to a salaried status of doctors in the hospital organization. The Dutch experience of the change of the payment system for medical specialists illustrates the transition to a new 'negotiated order'. The introduction of the 'lump sum' and the sub-contractor relationship with the insurance companies leaves the organizational autonomy of medical specialists intact. In exchange the medical specialists cooperate with the insurers in trying to control the costs of health care. In this process of strategic change, two factors are very significant, i.e. the new leadership of the local medical specialists and the governmental 'circumvention' of the powerful associations of doctors and insurers.

Exocytotic release of vasoative intestinal polypeptide and serotonin from mucosal nerve fibres and endocrine cells of the intestine of the goldfish (Carassius auratus) and the tilapia (Oreochromis mossambicus): an ultrastructural study.

In earlier studies were determined the effect, presence and ultrastructure of vasoactive intestinal polypeptide (VIP) and 5-hydroxytryptamine (5-HT)-containing nerve fibres in the tilapia and goldfish intestinal mucosa. 5-HT-labelled varicosities were found close to the epithelial cells; however, synaptic membrane specializations have never been observed. VIP-like immunoreactive nerve fibres appear to be located less frequently close to the goldfish epithelium, as in the tilapia intestine, in which the distance between the VIP- or 5-HT-labelled varicosities and the epithelial cells was also rather large (more than 2 micros). To establish a possible role of VIP and 5-HT as neurotransmitters involved in the regulation of fish intestinal epithelium both electron microscopical and immunoelectron microscopical methods were used to visualize the release of 5-HT and VIP from nerve fibres. We found exocytoses from VIP-ergic and serotonergic varicosities in the muscle layers of both fish. Directly underneath the intestinal epithelium of the goldfish, it was demonstrated that 5-HT could be released from scarce varicosities. The release of 5-HT in the tilapia intestinal mucosa could only be observed from endocrine cells.

Influence of forskolin and carbachol on intestinal absorption of horseradish peroxidase in the goldfish (Carassius auratus).

The transepithelial route for mucosa-to-serosa transport of the tracer macromolecule horseradish peroxidase (HRP; MW 40 kDa) and modulation of this transport by forskolin and carbachol have been studied in vi-tro in stripped goldfish intestinal epithelium mounted in Ussing-type chambers. Uptake and transport have been investigated by measuring the HRP flux from the muco-sal to serosal sides by an enzymatic method and by visualising HRP reaction products in the mucosa with electron-microscopical techniques. Both the cholinergic agonist carbachol (which is thought to increase intracellular Ca2+ and activate protein kinase C activity) and forskolin (a direct activator of adenylylcyclase) affect the amount of enzymatically active HRP in the tissue. In control tissue, HRP product is found only within the epithelial cells, the transepithelial flux reaching a constant value of about 1.5 pmoles/cm2 per h. Carbachol increases the amount of HRP product in the cells, but has no significant effect on the HRP flux compared with control values. Forskolin decreases the amount of HRP product in the cells; however, in the presence of forskolin, the lateral intercellular spaces become filled with HRP product. HRP is found in the lamina propria and the transepithelial protein flux increases more than 2.5-fold. In the presence of forskolin plus carbachol, the results are no different from the control. It is concluded that carbachol increases the endocytotic uptake of HRP, whereas forskolin inhibits the uptake but increases the paracellular permeability for HRP in goldfish intestine.

Carbachol, but not forskolin, increases mucosal-to-serosal transport of intact protein in rat ileum in vitro.

The effects of the secretagogues forskolin and carbachol on protein uptake in isolated ileum of rats were studied. The mucosal-to-serosal transport of horseradish peroxidase (HRP, mol mass 40 kDa) was measured in Ussing chambers, and afterwards tissues were processed for electron microscopy. In the absence of secretagogues, the flux of enzymatically active HRP was 5 pmol.cm-2.h-1 at a mucosal concentration of 10 microM. Electron micrographs showed vesicles filled with active HRP in enterocytes but no HRP activity in intercellular spaces. Forskolin decreased HRP activity in the cells. Carbachol increased the amount of HRP-filled vesicles in enterocytes and induced HRP filling in some intercellular spaces and tight junctions in the upper parts of the villi. The transepithelial flux of intact HRP increased more than 2.5-fold. This effect was suppressed by atropine. We conclude that cholinergic activation can increase the uptake of intact protein via endocytosis and the transepithelial passage by the induction of a diffusional paracellular pathway. We speculate that the increased transport of intact protein through the intestinal barrier may influence immunologic sensitization to food allergens.

Dopaminergic and GABAergic cerebrospinal fluid-contacting neurons along the central canal of the spinal cord of the eel and trout.

In anamniote vertebrates the central region of the spinal cord has been implicated in its regeneration. This is a complex region and so as a first step in understanding its possible regenerative role we have examined the organization of the cells that contact the lumen of the spinal cord in two teleost fishes, eel and trout, using immunohistochemical procedures and light and electron microscopy. Cell bodies immunoreacting positively with antibodies for tyrosine hydroxylase and for dopamine were located at the ventral rim of the central canal, whereas cell bodies reacting for an antibody for gamma-aminobutyric acid were more laterally located. None of the canal-contacting cells were positively immunoreactive for choline acetyltransferase. All immunopositive cells have a similar morphology: the amphora-shaped perikaryon is bipolar and has a single process that extends to the lumen of the canal, and another that branches and forms extensive lateral and ventral plexuses. Electron microscopic investigations of the ventral dopaminergic cells showed that the apical processes bear one or more cilia, which protrude into the canal lumen and which originate from within a superficial rosette of nonciliated processes. The ventral process was occasionally seen to form synapses; the cell body was also the target of synapses.

GABAA receptor function in the early period after transient forebrain ischaemia in the rat.

The purpose of this study was to evaluate the function of the GABAA receptor following transient forebrain ischaemia. The GABA-stimulated chloride (36Cl-) uptake into synaptoneurosomes was determined as an indicator of GABAA receptor function. Synaptoneurosomes were isolated from control rats and rats in which the forebrain was made ischaemic by way of the two-vessel occlusion model. Animals subjected to ischaemia were killed at the end of the ischaemic insult and at 30 min or 2 or 5 h of recirculation. The results showed a reduction of 75% in GABA-mediated 36Cl- uptake in synaptoneurosomes isolated from animals shortly (< 0.5 h) after the ischaemic episode (P < 0.01). After longer recirculation periods the GABA-mediated 36Cl- uptake reached preischaemic control levels. To investigate whether alterations in 36Cl- uptake were related to the synaptoneurosomal metabolic status, the synaptoneurosomal ATP content was measured. The time course of the ATP recovery correlated with the recovery of the GABA-mediated 36Cl- uptake (r = 0.7, P < 0.001). To investigate the importance of ATP in GABA-mediated 36Cl- uptake more directly, synaptoneurosomes isolated from control rats were exposed to chemically induced ATP depletion with rotenone, an inhibitor of oxidative phosphorylation. This resulted in similar reductions in both ATP level and GABA-stimulated 36Cl- uptake as observed after in vivo ischaemia. These findings indicate that GABAA receptor function is transiently impaired in the early postischaemic period in a way which is closely related to alterations in cellular energy metabolism. The relevance of these findings to the development of ischaemic cell death is discussed.

Differential release of amino acids, neuropeptides, and catecholamines from isolated nerve terminals.

We have investigated transmitter release from small and large dense-core vesicles in nerve terminals isolated from guinea pig hippocampus. Small vesicles are found in clusters near the active zone, and large dense-core vesicles are located at ectopic sites. The abilities of Ca2+ channel activation and uniform elevation of Ca2+ concentration (with ionophores) to evoke secretion of representative amino acids, catecholamines, and neuropeptides were compared. For a given increase in Ca2+ concentration, ionophore was less effective than Ca2+ channel activation in releasing amino acids, but not in releasing cholecystokinin-8. Titration of the average Ca2+ concentration showed that the Ca2+ affinity for cholecystokinin-8 secretion was higher than that for amino acids. Catecholamine release showed intermediate behavior. It is concluded that neuropeptide release is triggered by small elevations in the Ca2+ concentration in the bulk cytoplasm, whereas secretion of amino acids requires higher elevations, as produced in the vicinity of Ca2+ channels.

Myofibrillar differences among mammalian skeletal muscle fibres at the ultrastructural level. A comparison of immunocytochemical and morphometrical parameters.

In the light microscope two types (I, II) of skeletal muscle fibres can be distinguished with antibodies against myosin isozymes. At the ultrastructural level a difference in Z-line width has led to muscle fibre classification. In this study we distinguish at the ultrastructural level between type I and type II fibres of the M. soleus and M. plantaris of adult mice using ultracryosections with immuno-ferritin and antisera against myosin isozymes. Muscle fibres of the M. plantaris are identified as type II fibres and the fibres of the M. soleus are divided in type I and type II fibres. In the immunologically identified fibres the filament overlap in the Z-line was measured. The type II fibres of the M. plantaris have narrow Z-lines, whereas type I and type II fibres of the M. soleus have wide Z-lines. We conclude that a classification of fibres based on Z-line width differs from the type I/type II classification. The antimyosin antibodies react exclusively with the A-band. In serial sections the myosin isozymes can be identified unambiguously. This is a prerequisite for further studies of myosin isozyme distribution in "mixed" muscle fibres.

Quantitative succinate-dehydrogenase histochemistry. I. A Methodological study on mammalian and fish muscle.

In enzyme histochemistry formazan production can be used as a measure for oxidative enzyme activity. The formazan deposits can be measured quantitatively per cell with a scanning and integrating microspectrophotometer. Optimal conditions are described for the estimation of histochemical succinate dehydrogenase activity in sections of fish bodymusculature and mouse soleus and plantaris muscle. It is shown that when proper measuring conditions are chosen a ditetrazolium salt (TNBT) can be used in quantitative enzyme histochemistry and that the optimal conditions for the histochemical succinate dehydrogenase reaction in muscle fibres of fish and mouse muscle are somewhat different for these two species. The differences in pH, temperature and succinate sensitivity are the most prominent.