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Endocytosis and targeting of growth factor receptors for lysosomal degradation have been associated with ubiquitination of the intracellular part of the receptors. To elucidate the role of receptor ubiquitination in internalization and sorting of fibroblast growth factor receptor (FGFR), we constructed several mutants of FGFR1 in which lysines, potential ubiquitination sites, were substituted for arginines. Substitution of all lysine residues in the intracellular part of FGFR1 resulted in inactivation of the tyrosine kinase domain of the receptor. However, several multilysine FGFR1 mutants, where up to 26 of 29 lysines in the intracellular part of the receptor were mutated, retained tyrosine kinase activity. The active multilysine mutants were poorly ubiquitinated, but internalized normally, indicating that ubiquitination of the receptor is not required for endocytosis. In contrast, degradation of the multilysine mutants was dramatically reduced as the mutants were inefficiently transported to lysosomes but rather sorted to recycling endosomes. The altered sorting resulted in sustained signaling. The duration of FGFR1 signaling seems to be tightly regulated by receptor ubiquitination and subsequent sorting to the lysosomes for degradation.

Receptor-bound and endocytosed fibroblast growth factor-1 (FGF-1) is able to cross the vesicle membrane and translocate to cytosol and nucleus. This suggests an intracellular role of FGF-1, which also signals by activating transmembrane FGF receptors. Phosphorylation of internalized FGF-1 by nuclear protein kinase C delta induces rapid export from the nuclei by a leptomycin B-sensitive pathway. In the present work, we have searched for and identified a Leu-rich nuclear export sequence (NES) at the C terminus of FGF-1 required for its nuclear export and able to confer nuclear export activity to a reporter protein in an in vivo system. Mutants where hydrophobic amino acids within the NES were exchanged for alanine exhibited reduced or abolished nuclear export. As demonstrated in co-immunoprecipitation experiments, a complex containing FGF-1, exportin-1, and its co-factor Ran-GTP, was formed in vitro. Formation of this complex in vivo was demonstrated by a peroxisomal targeting assay. Formation of the FGF-1-exportin-1-Ran-GTP complex in vitro as well as nuclear export of FGF-1 in vivo was dependent on phosphorylation of FGF-1, and it was abolished by leptomycin B. The FGF-1 NES was found to be situated along a beta-strand, which has not been reported before, since NESs usually are alpha-helical.

Members of the fibroblast growth factor family bind to one or more of the four closely related membrane-spanning FGF receptors. In addition to signaling through the receptors, exogenous FGF-1 and FGF-2 are endocytosed and translocated to the cytosol and nucleus where they stimulate RNA and DNA synthesis. Here we have studied the ability of the four FGF receptors to facilitate translocation of exogenous FGF-1 to the cytosol and nucleus. FGFR1 and FGFR4 were able to mediate translocation, whereas FGFR2 and FGFR3 completely lacked this ability. By analyzing mutant FGFRs we found that the tyrosine kinase domain could be deleted from FGFR1 without abolishing translocation, whereas the C-terminal tail of the FGFRs, constituted by approximately 50 amino acids downstream of the kinase domain, plays a crucial role in FGF-1 translocation. Three amino acids residues within the C-terminal tail were found to be of particular importance for translocation. For FGFR2, the two amino acid substitutions Q774M and P800H were sufficient to enable the receptor to support FGF-1 translocation. The results demonstrate a striking diversity in function of the four FGFRs determined by their C-terminal domain.

Similarly to many protein toxins, the growth factors fibroblast growth factor 1 (FGF-1) and FGF-2 translocate from endosomes into the cytosol. It was recently found that certain toxins are dependent on cytosolic Hsp90 for efficient translocation across the endosomal membrane. We therefore investigated the requirement for Hsp90 in FGF translocation. We found that low concentrations of the specific Hsp90 inhibitors, geldanamycin and radicicol, completely blocked the translocation of FGF-1 and FGF-2 to the cytosol and the nucleus. The drugs did not interfere with the initial binding of FGF-1 to the growth factor receptors at the cell-surface or with the subsequent internalization of the growth factors into endosomes. The activation of known signaling cascades downstream of the growth factor receptors was also not affected by the drugs. The data indicate that the drugs block translocation from endosomes to the cytosol implying that Hsp90 is required for translocation of FGF-1 and FGF-2 across the endosomal membrane.

Many growth factors and cytokines bind to more than one receptor, but in many cases the different roles of the separate receptors in signal transduction are unclear. Intracellular sorting of ligand-receptor complexes may modulate the signalling, and we have here studied the intracellular trafficking of ligand bound to receptors for fibroblast growth factors (FGFs). For this purpose, we transfected HeLa cells with any one of the four tyrosine kinase FGF receptors (FGFR1-4). In cells expressing any one of these receptors, externally added FGF1 was localized to sorting/early endosomes after 15 minutes at 37 degrees C. After longer incubation times, FGF1 internalized in cells expressing FGFR1 was localized mainly to late endosomes/lysosomes, similarly to EGF. By contrast, FGF1 internalized in cells expressing FGFR4 followed largely the same intracellular pathway as the recycling ligand, transferrin. In cells expressing FGFR2 or FGFR3, sorting of FGF1 to lysosomes was somewhat less efficient than that observed for FGFR1. Furthermore, FGF1 was more slowly degraded in cells expressing FGFR4 than in cells expressing FGFR1-3 and in addition, internalized FGFR4 as such was more slowly degraded than the other receptors. The data indicate that after endocytosis, FGFR4 and its bound ligand are sorted mainly to the recycling compartment, whereas FGFR1-3 with ligand are sorted mainly to degradation in the lysosomes. Alignment of the amino acid sequence of the intracellular part of the four FGFRs revealed several lysines conserved in FGFR1-3 but absent in FGFR4. Lysines are potential ubiquitylation sites and could thus target a receptor to lysosomes for degradation. Indeed, we found that FGFR4 is less ubiquitylated than FGFR1, which could be the reason for the different sorting of the receptors.

Externally added FGF-1 is transported into the nucleus of cells. It was earlier shown that FGF-1 contains an N-terminal nuclear localization signal (NLS) implicated in the stimulation of DNA synthesis. We here provide evidence that FGF-1 contains a second putative NLS (NLS2), which is located near the C-terminus. It is a bipartite NLS consisting of two clusters of lysines separated by a spacer of 10 amino acids. A fusion protein of GFP and the bipartite NLS was more efficiently transported into the nucleus than GFP alone, indicating that it can act as an NLS in the living cell. FGF-1 mutated in the N-terminal NLS (NLS1) or in the first cluster of the bipartite NLS2 bound to heparin and FGF receptors and activated downstream signaling similarly to the wild-type growth factor. Mutations in the second cluster of NLS2 resulted in impaired interaction with heparin and reduced stability. When radiolabeled FGF-1 with mutated NLS1 or the first lysine cluster of NLS2 was added to NIH/3T3 cells, it was translocated into the cytosol, but not transported efficiently to the nucleus. Phosphorylation of FGF-1 occurs normally in the nucleus, and while wild-type FGF-1 was phosphorylated after addition to cells, the NLS mutants were not. It therefore appears that both NLS1 and NLS2 are important for efficient transport of FGF-1 to the nucleus. Stimulation of DNA synthesis by FGF-1 with mutations in both NLSs was reduced considerably indicating that efficient transport to the nucleus may be involved in the stimulation of DNA synthesis.

Fibroblast growth factor-1 (FGF-1), which stimulates cell growth, differentiation, and migration, is capable of crossing cellular membranes to reach the cytosol and the nucleus in cells containing specific FGF receptors. The cell entry process can be monitored by phosphorylation of the translocated FGF-1. We present evidence that phosphorylation of FGF-1 occurs in the nucleus by protein kinase C (PKC)delta. The phosphorylated FGF-1 is subsequently exported to the cytosol. A mutant growth factor where serine at the phosphorylation site is exchanged with glutamic acid, to mimic phosphorylated FGF-1, is constitutively transported to the cytosol, whereas a mutant containing alanine at this site remains in the nucleus. The export can be blocked by leptomycin B, indicating active and receptor-mediated nuclear export of FGF-1. Thapsigargin, but not leptomycin B, prevents the appearance of active PKCdelta in the nucleus, and FGF-1 is in this case phosphorylated in the cytosol. Leptomycin B increases the amount of phosphorylated FGF-1 in the cells by preventing dephosphorylation of the growth factor, which seems to occur more rapidly in the cytoplasm than in the nucleus. The nucleocytoplasmic trafficking of the phosphorylated growth factor is likely to play a role in the activity of internalized FGF-1.

The entry of exogenous fibroblast growth factor 2 (FGF-2) to the cytosolic/nuclear compartment was studied and compared with the translocation mechanism used by FGF-1. To differentiate between external and endogenous growth factor, we used FGF-2 modified to contain a farnesylation signal, a CaaX-box. Because farnesylation occurs only in the cytosol and nucleoplasm, farnesylation of exogenous FGF-2-CaaX was taken as evidence that the growth factor had translocated across cellular membranes. We found that FGF-2 translocation occurred in endothelial cells and fibroblasts, which express FGF receptors, and that the efficiency of translocation was increased in the presence of heparin. Concomitantly with translocation, the 18-kDa FGF-2 was N-terminally cleaved to yield a 16-kDa form. Translocation of FGF-2 required PI3-kinase activity but not transport through the Golgi apparatus. Inhibition of endosomal acidification did not prevent translocation, whereas dissipation of the vesicular membrane potential completely blocked it. The data indicate that translocation occurs from intracellular vesicles containing proton pumps and that an electrical potential across the vesicle membrane is required. Translocation of both FGF-1 and FGF-2 occurred during most of G(1) but decreased shortly before the G(1)-->S transition. A common mechanism for FGF-1 and FGF-2 translocation into cells is postulated.

In this paper we report on searching for suitable reporters to monitor gene expression and protein secretion in the amylolytic yeast Schwanniomyces occidentalis. Several potential reporter and marker genes, formerly shown to be functional in other yeasts, were cloned downstream from the homologous invertase gene (INV) promoter and their activity was followed in conditions of repression and derepression of the INV promoter. However, neither beta-glucuronidase nor beta-lactamase nor phleomycin resistance-conferring gene, all originating from E. coli, were expressed in S. occidentalis cells to such a level to allow for monitoring of their activity. All the reporter genes tested have a higher percentage of GC (47-62%) in their DNA compared to the DNA composition of S. occidentalis genes that are more AT-rich (36% GC). The codon usage of all the reporter genes also varies from that of 16 so far sequenced S. occidentalis genes. This suggests that an appropriate composition of DNA and a codon usage similar to S. occidentalis genes might be very important parameters for an efficient expression of a heterologous gene in Schwanniomyces occidentalis. Indeed, two genes originating from Staphylococcus aureus, with an AT-content in their DNA similar to that of S. occidentalis, were functionally expressed in S. occidentalis cells. Both a phleomycin resistance-conferring gene and a chloramphenicol acetyltransferase-encoding gene thus represent suitable reporters of gene expression and protein secretion in S. occidentalis. Additionally, we show in this work that the transcription-regulating region and the signal peptide sequence of the S. occidentalis invertase gene were efficient to direct gene expression and subsequent protein secretion in Saccharomyces cerevisiae., (Copyright 2003 John Wiley & Sons, Ltd.)

Externally added fibroblast growth factor-1 (FGF-1) is capable of crossing cellular membranes to reach the cytosol and the nucleus in a number of cell types. We have monitored the translocation of the growth factor by two methods: phosphorylation of FGF-1, and prenylation of an FGF-1 mutant that contains a C-terminal prenylation signal. Inhibition of endosomal acidification by ammonium chloride or monensin did not block the translocation of FGF-1, whereas bafilomycin A1, a specific inhibitor of vacuolar proton pumps, blocked translocation completely. A combination of ionophores expected to dissipate the vesicular membrane potential (valinomycin plus monensin) also fully inhibited the translocation. The inhibition of translocation by bafilomycin A1 was overcome in the presence of monensin or nigericin, while ouabain blocked translocation under these conditions. The data indicate that translocation of FGF-1 to cytosol occurs from the lumen of intracellular vesicles possessing vacuolar proton pumps, and that a vesicular membrane potential is required. Apparently, activation of vesicular Na+/K+-ATPase by monensin or nigericin generates a membrane potential that can support translocation when the proton pump is blocked.

Fibroblast growth factor-1 (FGF-1) has both extra- and intracellular functions. To identify intracellular binding partners for FGF-1, we isolated proteins from U2OS human osteosarcoma cells interacting specifically with FGF-1. One of the isolated proteins was identified as protein kinase CK2 (CK2). We here provide evidence that FGF-1 binds to both the catalytic alpha-subunit and to the regulatory beta-subunit of CK2. The interaction between FGF-1 and CK2 alpha and beta was characterized by surface plasmon resonance, giving K(D) values of 0.4 +/- 0.3 and 1.2 +/- 0.2 microM, respectively. By using a novel assay for intracellular protein interaction, FGF-1 and CK2 alpha are shown to interact in vivo. In vitro, FGF-1 and FGF-2 are phosphorylated by CK2, and the presence of FGF-1 or FGF-2 was found to enhance the autophosphorylation of CK2 beta. A correlation between the mitogenic potential of FGF-1 mutants and their ability to bind to CK2 alpha was observed. The possible involvement of CK2 in the FGF-induced stimulation of DNA synthesis is discussed.

With the aim of identifying new intracellular binding partners for acidic fibroblast growth factor (aFGF), proteins from U2OS human osteosarcoma cells were adsorbed to immobilized aFGF. One of the adsorbed proteins is a member of the leucine-rich repeat protein family termed ribosome-binding protein p34 (p34). This protein has previously been localized to endoplasmic reticulum membranes and is thought to span the membrane with the N terminus on the cytosolic side. Confocal microscopy of cells transfected with Myc-p34 confirmed the endoplasmic reticulum localization, and Northern blotting determined p34 mRNA to be present in a multitude of different tissues. Cross-linking experiments indicated that the protein is present in the cell as a dimer. In vitro translated p34 was found to interact with maltose-binding protein-aFGF through its cytosolic coiled-coil domain. The interaction between aFGF and p34 was further characterized by surface plasmon resonance, giving a K(D) of 1.4 +/- 0.3 microm. Even though p34 interacted with mitogenic aFGF, it bound poorly to the non-mitogenic aFGF(K132E) mutant, indicating a possible involvement of p34 in intracellular signaling by aFGF.

The plant toxin ricin binds to terminal galactose-containing cell-surface receptors. The toxin is endocytosed and transported to the Golgi apparatus. Recent evidence suggests that ricin binds to galactosylated calreticulin, which may carry the toxin from the Golgi apparatus to the endoplasmic reticulum (ER). From the ER, the ricin A fragment is translocated to the cytosol. Ricin is perceived to be a candidate for ER-associated degradation (ERAD) and is translocated through the Sec61p translocon to the cytosol. Part of the toxin is degraded by the proteasome, but a fraction of the ricin avoids degradation and inhibits protein synthesis by inactivating ribosomes, ultimately leading to cell death.

Endocytic uptake and intracellular transport of acidic fibroblast growth factor (aFGF) was studied in cells transfected with FGF receptor 4 with mutations in the cytoplasmic part. Endocytic uptake in HeLa cells was reduced but not abolished when the tyrosine kinase of the receptor was inactivated by mutations or deletions. The tyrosine kinase-dependent endocytosis of aFGF was prevented by the expression of a dominant negative dynamin mutant that blocks endocytosis from coated pits and caveolae. However, more than half of the total endocytic uptake of aFGF was not affected under these conditions, indicating an endocytic uptake mechanism not involving coated pits or caveolae. Mutation or deletion of a putative caveolin-binding sequence did not prevent the localization of part of the receptors to a low density, caveolin-containing subcellular fraction. Whereas wild-type receptor transfers the growth factor from early endosomes to the recycling compartment, kinase negative, full length receptors were inefficient in this respect and the growth factor instead accumulated in lysosomes. By contrast, when most of the intracellular part of the receptor, including the kinase domain, was removed, aFGF was transported to the recycling compartment, as in cells that express wild-type receptors, suggesting the presence of a kinase-regulated targeting signal in the cytoplasmic tail.

A number of proteins are able to enter cells from the extracellular environment, including protein toxins, growth factors, viral proteins, homeoproteins, and others. Many such translocating proteins, or parts of them, appear to be able to carry with them cargo into the cell, and a basic sequence from the HIV-1 Tat protein has been reported to provide intracellular delivery of several fused proteins. For evaluating the efficiency of translocation to the cytosol, this TAT-peptide was fused to the diphtheria toxin A-fragment (dtA), an extremely potent inhibitor of protein synthesis which normally is delivered efficiently to the cytosol by the toxin B-fragment. The fusion of the TAT-peptide to dtA converted the protein to a heparin-binding protein that bound avidly to the cell surface. However, no cytotoxicity of this protein was detected, indicating that the TAT-peptide is unable to efficiently deliver enzymatically active dtA to the cytosol. Interestingly, the fused TAT-peptide potentiated the binding and cytotoxic effect of the corresponding holotoxin. We made a fusion protein between VP22, another membrane-permeant protein, and dtA, and also in this case we detected association with cells in the absence of a cytotoxic effect. The data indicate that transport of dtA into the cell by the TAT-peptide and VP22 is inefficient.

Here we demonstrate that ricin is able to interact with the molecular chaperone calreticulin both in vitro and in vivo. The interaction occurred with ricin holotoxin, but not with free ricin A chain; and it was prevented in the presence of lactose, suggesting that it was mediated by the lectin activity of the ricin B chain. This lectin is galactose-specific, and metabolic labeling with [(3)H]galactose or treating galactose oxidase-modified calreticulin with sodium [(3)H]borohydride indicated that Vero cell calreticulin possesses a terminally galactosylated oligosaccharide. Brefeldin A treatment indicated that the intracellular interaction occurred initially in a post-Golgi stack compartment, possibly the trans-Golgi network, whereas the reductive separation of ricin subunits occurred in an earlier part of the secretory pathway, most probably the endoplasmic reticulum (ER). Intoxicating Vero cells with ricin whose A chain had been modified to include either a tyrosine sulfation site or the sulfation site plus available N-glycosylation sites, in the presence of Na(2)35SO(4), confirmed that calreticulin interacted with endocytosed ricin that had already undergone retrograde transport to both the Golgi and the ER. Although we cannot exclude the possibility that the interaction between ricin and calreticulin is an indirect one, the data presented are consistent with the idea that calreticulin may function as a recycling carrier for retrograde transport of ricin from the Golgi to the ER.

Acidic fibroblast growth factor (aFGF) is transported to the cytosol and the nucleus when added to cells expressing FGF receptors, implying that aFGF must cross cellular membranes. Since protein translocation across membranes commonly requires extensive unfolding of the protein, we were interested in testing whether this is also necessary for membrane translocation of aFGF. We therefore constructed mutant growth factors with intramolecular disulfide bonds to prevent complete unfolding. Control experiments demonstrated that translocation of aFGF by the diphtheria toxin pathway, which requires extensive unfolding of the protein, was prevented by disulfide bond formation, indicating that the introduced disulfide bonds interfered with the unfolding of the growth factor. On the other hand, when the growth factor as such was added to cells expressing FGF receptors, the disulfide-bonded mutants were translocated to the cytosol and the nucleus equally well as wild-type aFGF. The possibility that the translocation of the mutants was due to reduction of the disulfide bonds prior to translocation was tested in experiments using an irreversibly cross-linked mutant. Also this mutant was transported to the cytosol and to the nucleus. The results suggest that extensive unfolding is not required for membrane translocation of aFGF.

Endocytic uptake and intracellular transport of acidic FGF was studied in cells transfected with FGF receptor 4 (FGFR4). Acidification of the cytosol to block endocytic uptake from coated pits did not inhibit endocytosis of the growth factor in COS cells transfected with FGFR4, indicating that it is to a large extent taken up by an alternative endocytic pathway. Fractionation of the cells demonstrated that part of the growth factor receptor was present in a low-density, caveolin-containing fraction, but we were unable to demonstrate binding to caveolin in immunoprecipitation studies. Upon treatment of the cells with acidic FGF, the activated receptor, together with the growth factor, moved to a juxtanuclear compartment, which was identified as the recycling endosome compartment. When the cells were lysed with Triton X-100, 3-([3-chloramidopropyl]dimethylammonio)-2-hydroxy-1-propanesulfona te, or 2-octyl glucoside, almost all surface-exposed and endocytosed FGFR4 was solubilized, but only a minor fraction of the total FGFR4 in the cells was found in the soluble fraction. The data indicate that the major part of FGFR4 is anchored to detergent-insoluble structures, presumably cytoskeletal elements associated with the recycling endosome compartment.

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Solving the crystallographic structure of the ring-shaped heptamer formed by protective antigen (PA), the B moiety of anthrax toxin, has focused attention on understanding how this oligomer mediates membrane translocation of the toxin's A moieties. We have developed an assay for translocation in which radiolabeled ligands are bound to proteolytically activated PA (PA63) at the surface of CHO or L6 cells, and translocation across the plasma membrane is induced by lowering the pH. The cells are then treated with Pronase E to degrade residual surface-bound material, and protected ligands are quantified after fractionation by SDS-PAGE. Translocation was most efficient (35%-50%) with LFN, the N-terminal PA binding domain of the anthrax lethal factor (LF). Intact LF, edema factor (EF), or fusion proteins containing LFN fused to certain heterologous proteins [the diphtheria toxin A chain (DTA) or dihydrofolate reductase (DHFR)] were less efficiently translocated (15%-20%); and LFN fusions to several other proteins were not translocated at all. LFN with different N-terminal residues was found to be degraded according to the N-end rule by the proteasome, and translocation of LFN fused to a mutant form of DHFR with a low affinity for methotrexate (MTX) protected cells from the effects of MTX. Both results are consistent with a cytosolic location of protected proteins. Evidence that a protein must unfold to be translocated was obtained in experiments showing that (i) translocation of LFNDTA was blocked by introduction of an artificial disulfide into the DTA moiety, and (ii) translocation of LFNDHFR and LFNDTA was blocked by their ligands (MTX and adenine, respectively). These results demonstrate that the acid-induced translocation by anthrax toxin closely resembles that of diphtheria toxin, despite the fact that these two toxins are unrelated and form pores by different mechanisms.

Beat-by-beat heart rate (HR) changes during exercise were studied in two young and fit heart-transplanted humans at different time intervals following transplantation. Upon the start of the exercise, a slow gradual increase in HR was seen during the early experiments after the transplantation, whereas an immediate rapid increase in HR was observed during the later experiments. From standard ECGs obtained 32 months after transplantation, two P waves at somewhat different rates could be identified in both subjects, probably arising from donor and recipient sinoatrial nodes, respectively. The two P wave rate changes during and following exercise were very similar. We conclude that these changes in the HR pattern and ECG must be due to reinnervation of the donor hearts, most likely by parasympathetic cardiac fibers.

The haemodynamic effects of 45 min of treadmill exercise (at 70% of resting heart rate reserve) were determined in 5 young adults with hypertension and rapid resting heart rates (greater than 90 beats/min in clinic) and were compared with those of 5 age-matched normotensive subjects. Blood pressure was lower after exercise in the hypertensive, but not the normotensive subjects. Mean cardiac output before exercise was similar in the two groups, and fell from 6.8 +/- 0.6 before to 5.4 +/- 0.6 l/min 60 min after exercise in the hypertensive group (P less than 0.01). Total peripheral resistance tended to be higher at this time. Neither variable was affected by prior exercise in the normotensive group. The depressor effects of prior exercise on mean arterial pressure (-8.6 +/- 1.0 vs. -1.4 +/- 2.5 mmHg; P less than 0.04) and cardiac output (-1.4 +/- 0.3 vs. -0.1 +/- 0.1 l/min; P less than 0.005) and the increase in total peripheral resistance (+3.0 +/- 1.2 vs. 0.0 +/- 1.0 Units; P less than 0.05) were greater in the hypertensive group. Thus, the post-exercise hypotension in this selected group of young hypertensive subjects with rapid resting heart rates was mediated by a decrease in cardiac output and stroke volume disproportionate to the fall in blood pressure, suggesting sustained compromise of their cardiac performance after acute exercise.

A pulsed Doppler ultrasound velocity meter was used to measure blood velocities in the right uterine artery and a mammary branch of the right lateral thoracic artery in one subject throughout pregnancy and after parturition. The breast blood velocity was also followed throughout lactation. The velocities in the uterine artery increased 2.5-fold from the 15th to the 38th week, when delivery occurred. An estimate of the total blood flow in the uterine artery, based on the total intensities in the Doppler spectrum, increased 5-fold in the same period. This corresponds to the increase in volume flow obtained with invasive methods. Velocities in the breast artery increased 2.5-fold from the 12th to the 25th week of pregnancy and then stayed at the same level throughout lactation; after weaning, they rapidly declined.

A heterodyne pulsed doppler velocitymeter was used to measure blood velocities in the mammary branch of the lateral thoracic artery during breast-feeding and after oxytocin injections. A few heart beats before the mother felt the milk-ejection reflex, blood velocities fell rapidly by 40-50%, and then increased during the next 1-2 min. Synthetic oxytocin was given intravenously to lactating women, both in doses believed to be physiological in milk-ejection and larger, and the same velocity changes occurred. Synthetic oxytocin was also given to non-lactating women who had never been pregnant or who had breast-fed previously and to one man. In these a rapid increase in velocity was observed after about a half a minute, lasting for 1-2 min. In addition to the short-term effects of breast-feeding, blood velocity increased rapidly towards the end of nursing or a few minutes afterwards, with a maximal increase of 25-50%. It then slowly returned to pre-nursing values during the following 30-60 min. This pattern was seen in both breasts, independent of which breast the infant had been sucking.