Molecular basis of human ATM kinase inhibition.

Human checkpoint kinase ataxia telangiectasia-mutated (ATM) plays a key role in initiation of the DNA damage response following DNA double-strand breaks. ATM inhibition is a promising approach in cancer therapy, but, so far, detailed insights into the binding modes of known ATM inhibitors have been hampered due to the lack of high-resolution ATM structures. Using cryo-EM, we have determined the structure of human ATM to an overall resolution sufficient to build a near-complete atomic model and identify two hitherto unknown zinc-binding motifs. We determined the structure of the kinase domain bound to ATPγS and to the ATM inhibitors KU-55933 and M4076 at 2.8 Å, 2.8 Å and 3.0 Å resolution, respectively. The mode of action and selectivity of the ATM inhibitors can be explained by structural comparison and provide a framework for structure-based drug design.

Astrocytes in sensory circumventricular organs of the rat brain express functional binding sites for endothelin.

Sensory circumventricular organs bordering the anterior third cerebral ventricle, the subfornical organ and the organum vasculosum laminae terminalis, lack blood-brain barrier characteristics and are therefore accessible to circulating peptides like endothelins. Astrocytes of the rat subfornical organ and organum vasculosum laminae terminalis additionally showed immunocytochemical localization of endothelin-1/endothelin-3-like peptides, possibly acting as circumventricular organ-intrinsic modulators. Employing [125I]endothelin-1 as radioligand, quantitative autoradiography demonstrated specific binding sites throughout the rat organum vasculosum laminae terminalis and subfornical organ, and competitive displacement studies revealed expression of both ET(A) and ET(B) receptor subtypes for either circumventricular organ. ET(B) receptor binding prevailed for the whole brain and ET(A) receptors could be labelled in the peripheral vascular system. To characterize endothelin-specific receptors in astrocytes of both circumventricular organs, alterations in the intracellular calcium concentration due to endothelin-1/endothelin-3 stimulation were studied in primary culture of subfornical organ and organum vasculosum laminae terminalis cells obtained from early postnatal rat pups. Endothelin-1 and endothelin-3 induced Ca(2+) transients in 9-13% of either subfornical organ or organum vasculosum laminae terminalis astrocytes, respectively, and some glial cells (subfornical organ: 2%, organum vasculosum laminae terminalis: 5%) responded to both endothelin analogues. The antagonistic action of BQ123 specific for ET(A) receptors (74% of all astrocytes tested), and the pronounced responsiveness to the ET(B) receptor agonist [4Ala]ET-1 (subfornical organ: 27%, organum vasculosum laminae terminalis: 35%) demonstrated glial expression of both endothelin receptor subtypes. Agonist-induced elevations in the intracellular calcium concentration proved to be independent of extracellular Ca(2+). In summary, the results indicate that endothelin(s) interact(s) with circumventricular organ astrocytes. Competitive receptor binding techniques using brain tissue sections as well as a fura-2 loaded primary cell culture system of the subfornical organ and organum vasculosum laminae terminalis demonstrate glial expression of functional ET(A) and ET(B) receptors, with calcium as intracellular messenger emerging primarily from intracellular stores. Endothelin(s) of both circulating and circumventricular organ-intrinsic origin may afferently transfer information important for cardiovascular homeostasis to circumventricular organs serving as "windows to the brain".

Spinal neuronal thermosensitivity in vivo and in vitro in relation to hypothalamic neuronal thermosensitivity.

In the spinal cord, temperature signals are generated which serve as specific inputs in the central nervous control of body temperature. Because of the spatially distinct organization of afferent and efferent neuronal systems at the spinal level, the afferent pathway for temperature signal transmission could be identified in vivo in the ascending, anterior and lateral tracts with a relationship of about 75:25% between warm and cold sensitive neuraxons. Analysis of spinal neuronal thermosensitivity in vitro on spinal cord tissue slices has been concerned, so far, with the superficial laminae of the dorsal horn as the site of origin of ascending nerve fibers conveying mostly temperature and pain signals, and with lamina X as a site of origin of afferent as well as efferent neurons. A relationship of about 95:5% between warm and cold sensitive neurons was found at the segmental level, indicating that warm sensitivity is the prevailing, primary property of spinal neurons, whereas cold sensitivity seems to be mainly generated by synaptic interaction as a secondary modality. Dynamic responses to temperature changes were frequently displayed in vitro at the spinal segmental level in lamina I + II but not in lamina X, even by neurons whose static activity was little influenced by local temperature. Dynamic thermosensitivity was found less frequently in ascending tract neuraxons and was not observed in hypothalamic neurons receiving temperature signal inputs from the spinal cord, and thus, does not seem to be relevant for the thermosensory function of spinal cord neurons, unlike peripheral warm and cold receptors. A majority of spinal warm sensitive neurons displayed both static and dynamic warm sensitivity as an inherent property after synaptic blockade. In the further analysis of spinal cord thermosensitivity, the in vitro approach permits application of the same electrophysiological and neuropharmacological methods as were established for the analysis of hypothalamic thermosensitivity. In addition, the topography of the spinal cord will provide additional structural and possibly histochemical information to characterize the functions of neurons independently of their thermal properties.

Electrophysiological responses of neurons in the rat spinal cord to nitric oxide.

The effects of nitric oxide-containing solution and different nitric oxide donors were investigated on spontaneously active neurons using extracellular recording technique in areas of rat spinal cord slices where high levels of nitric oxide synthase are present. In lamina X, 93% of all neurons investigated (n = 84) increased their firing rate and 2% decreased it by superfusion with the nitric oxide donor sodium nitroprusside. In contrast, 49% of all neurons in laminae I and II (n = 90) were inhibited and only 28% were activated. Both effects were due to the postsynaptic action of sodium nitroprusside, because they could still be observed in medium containing 0.3 mM Ca2+ and 9 mM Mg2+, known to block synaptic transmission. Application of 8-bromo-cyclic-GMP caused an excitation of every neuron which was excited by sodium nitroprusside and an inhibition of every cell which was inhibited by sodium nitroprusside (n = 25). This effect was different from the effect of 8-bromo-cyclic-AMP, which mimicked only the excitatory, but not the inhibitory response of sodium nitroprusside. These results provide evidence that nitric oxide in the spinal cord can directly cause an excitation or an inhibition of the electrical activity of spinal neurons. Another, more general conclusion from our results is that the nitric oxide-induced production of cyclic-GMP alone does not allow any prediction about an excitatory or inhibitory effect on the neuronal activity, which has to be determined separately.

Temperature sensitivity of neurones in slices of the rat spinal cord.

1. The inherent temperature sensitivity of 343 spontaneously active neurones recorded from rat spinal cord (SC) slices was investigated electrophysiologically. Recordings were made from 321 neurons from transverse and 22 neurons from longitudinal slices and their thermosensitivity was determined by relating changes in firing rate to changes in slice temperature. 2. Of the neurones from transverse slices, 53% were warm sensitive, 2% were cold sensitive and 45% were temperature insensitive. In longitudinal slices, 68% were warm sensitive and the remaining neurones were temperature insensitive. 3. When classified according to their recording sites in transverse slices, warm-sensitive neurones in laminae I and II had the same mean temperature coefficient compared with those recorded from lamina X, despite the fact that the latter had a significantly higher spontaneous activity. 4. The intrinsic temperature sensitivity of the majority of warm-sensitive neurones was confirmed by blocking their synaptic input. 5. A transient overshoot in activity, i.e. a dynamic response characteristic following rapid temperature stimuli (0.4 degree C s-1) was observed in 73% of the warm-sensitive and 59% of the temperature-insensitive neurones in laminae I and II in response to rapid warming, but only rarely (< 10%) in lamina X. 6. Temperature-sensitive SC neurones share response characteristics with temperature-sensitive neurones in the preoptic and anterior hypothalamic (PO/AH) area and with peripheral temperature receptors. Functionally, these neurones may represent the cellular basis for the temperature sensory function of the spinal cord that has been well characterized in vivo in homeothermic species.

Regional specific effects of nitric oxide donors and cGMP on the electrical activity of neurons in the rat spinal cord.

Numerous functional studies establish the role of nitric oxide (NO) as a neuromodulator in the central nervous system which affects synaptic transmission. However, there are only a few reports indicating a direct and postsynaptic effect of nitric oxide on the electrical activity of neurons in the central nervous system. The aim of this study was to characterize the effect of nitric oxide on spontaneously active neurons in spinal cord slices using an extracellular recording technique. Because in the lumbar rat spinal cord the NO producing enzyme NO-synthase is primarily located in the superficial dorsal horn (laminae I+II) and around the central canal (lamina X), we restricted our recordings to these areas. While the majority of neurons increased their electrical activity during superfusion with the NO-donor sodium nitroprusside (SNP) in lamina X, neurons in laminae I+II were mainly inhibited by SNP. The excitatory and the inhibitory effects were dose-dependent and reversible and were mimicked by other NO-donors and membrane permeable cyclic guanosine monophosphate (8Br-cGMP) on the same neurons. The spinal cord slice preparation contains functional NO-synthase (NOS), because selective blockade of NOS increased the spontaneous activity of those neurons from laminae I+II which were inhibited by SNP and this effect could be reversed by superfusion with the natural substrate for NOS, L-arginine. It is concluded that NO can activate and inhibit the activity of spinal cord neurons by raising cGMP levels and that these effects are lamina specific. A general consequence of our results is that the NO-induced production of cGMP alone does not allow any prediction about an excitatory or inhibitory effect of NO on the discharge rate of neurons. Thus the NO mediated increase and decrease in neuronal activity is probably the result of intracellular mechanisms downstream from the production of cGMP which results in the activation or inhibition of different ion channels on neurons in laminae I+II and X.